Renal Damage in the Mouse: The Response to Very Small Doses per Fraction

Author(s): M. C. Joiner and H. Johns

Source: Radiation Research, Vol. 114, No. 2 (May, 1988), pp. 385-398
Published by: Radiation Research Society
Stable URL: http://www.jstor.org/stable/3577233 .
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RADIATION 114, 385-398 (1988)
RESEARCH

RenalDamageintheMouse:The Responseto Very

SmallDoses perFraction

M. C. JOINERANDH. JOHNS

oftheCancerResearch
GrayLaboratory MountVernon
Campaign, Northwood,
Hospital,
MiddlesexHA62RN,UnitedKingdom

JOINER,M. C., ANDJOHNS,H. Renal Damage in the Mouse: The Response to VerySmall
Radiat.Res.114,385-398(1988).
DosesperFraction.

Experiments wereundertaken tostudy theeffectonthemousekidney ofrepeated X-raydoses

in therange0.2 to 1.6 Gy perfraction and neutron dosesin therange0.05 to 0.25 Gy per
fraction.A top-updesignofexperiment wasused,so thatadditional gradeddosesofd(4)-Be
neutrons (EN= 2.3 MeV)weregiventobring thesubthreshold damageproduced bythesetreat-
mentsintothemeasurable range.Thisapproach avoidedthenecessity tousea largenumber of
fractionstostudy lowdosesperfraction. Renaldamagewasassessedusingthreemethods: 51Cr-
EDTA clearance, urineoutput, andhematocrit at 16-50weekspostirradiation. The dose-re-
sponsecurvesobtained wereresolved bestat29 weeks.However, theresultswerealsoexamined
byfitting second-order polynomials to thedataforresponse versustimepostirradiation and
usinginterpolated valuesfrom thesefunctionsat 29 weeksto construct dose-response curves.
Thismethod reduced thevariation
slightly inthedose-response data,buttheinterrelationship
between thedose-response curvesremained thesame.The datawereusedto testthelinear-
quadratic oftheunderlying
(LQ) description X-raydose-fractionation relationship.Themodel
fitswelldowntoX-raydosesperfraction of- I Gy,butlowerX-raydosesweremoreeffective
pergraythanpredicted byLQ, as seenpreviously in skin[M. C. Joineretal., Int.J.Radiat.
Biol.49, 565-580(1986)].Thisincreased X-rayeffectiveness and deviation fromLQ arere-
flected
directly in a decreasein theRBE ofd(4)-Beneutrons relativeto X-raysat lowdoses,
sincetheunderlying response totheseneutrons is linearinthislow-dose region. TheRBE de-
from
creases 9.9to4.7as theX-ray doseperfraction isreducedbelow0.8Gyto0.2Gy,reflecting
an increasein X-rayeffectiveness bya factor of2.1. A modelis discussed whichattempts to
explainthisbehaviorat low doses perfraction. ? 1988Academic Inc.
Press,

INTRODUCTION

Fromthestudiesofnormaltissuesandtumors whichhavebeenreported so far,it

canbe concludedthatthedose-fractionation
response described
isalmostinvariably
bythewell-known
accurately (LQ) modelovertheradiation
linear-quadratic dose
rangefromlargesingledosesdowntodosesperfraction ofabout2 Gy.Thatthisone
simplemodelofradiation responsein vivohas beenfoundto be widelyapplicable
has improvedtheinterfaceofradiation and has
biologywithclinicalradiotherapy,
intopossiblewaysin whichradiationtreatment
givenfreshinsights mightbe im-
in vivohas also been supportedby
proved(1-8). The applicationofLQ relationships
biophysicalmodelswhichsuggesta morefundamentalbasis foran LQ relationship
385 $3.00
0033-7587/88
Copyright? 1988 byAcademic Press,Inc.
All rightsof reproductionin any formreserved.

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386 JOINERAND JOHNS

appliedto cellsurvival (e.g.,(9, 10)),although theactualrelationship between target

cellsurvivalandobserved functional deficitinvivois likelytobe complex(11,12).
theincreasingly
Clearly, wideuseoftheLQ modelmeansthatitslimitations must
be explored, andthecurrent interestin bothhyperfraction and accelerated fraction-
ation(e.g.,(13)) requiresthattheresponseoftissuesto lowdosesperfraction (<2
Gy)be examined. Thereis alreadyan indication thatdeviations fromtheLQ model
occuratsuchdoses(14-17).
Experiments in thisdoserangearedifficult to executebecausetheyrequirevery
manyfractions to be administered to achievea measurable endpoint.Forexample,
Stewart etal. (16) showthatitrequires atleast80 dosefractions tostudyX-raydoses
oflessthan0.8 Gyperfraction inmousekidney. Suchexperiments areusuallycom-
promised by the need for a short interval between fractions
to avoid excessivelylong
overalltimes,so thatrepairmaybe incomplete, andclearlybecomeimpractical ifthe
doseperfraction is reducedmuchfurther.
Theselogistical problems incarrying outsuchexperiments areovercome byusing
a top-upapproach(18),inwhicha reducednumber oflow-dose fractionsarefollowed
by an extra"top-up"dose ofneutrons to bringthesubthreshold damageintothe
measurable range. This experimental design also enablesthe effectofdifferent
doses
per fractionto be studied while maintaining a constant number of fractions,and
hencea constant overalltimeand constant interfraction
interval. Thesefactors are
thereforethesamein everytreatment, and so changesin effect reflectonlychanges
indoseperfraction, as required ina rigorous testoftheLQ model.
The experiments described inthispaperusethistop-uptechnique to examineX-
in
rayeffectivenessthemousekidney, usingdosesof0.2 to 1.6Gyperfraction. Ifthe
LQ modelholdsin thisdoseregion, a significant fractionationeffectwouldbe pre-
dictedforkidney(a/l 3 Gy),amounting to abouta 40% increasein thetotal
X-raydoseas thedoseperfraction
isoeffective wasreducedfrom1.6to 0.2 Gy.In
addition,we also comparedtheseX-raydatawiththeresultsfromsmallneutron
fractionsusing0.05to0.25Gyperfraction.

MATERIALSAND METHODS
Miceandirradiationschedule.FemaleCBA/Ht/GyfBSVS miceaged12-16weeks(19-25gweight) were
in airwithout
irradiated anesthetic. Six animalswereallocatedto eachdosegroupwhichreceived30
ofeither240 kVpX raysord(4)-Beneutrons
fractions in an overalltimeof 19days(3 working weeks),
usingtwofractionsperdaywithan interval of7 h and 10 fractionsperweek.Threedaysafterreceiving
their
lastradiation allmicereceived
fraction, a singletop-updoseofd(4)-Beneutrons, togivea totaloverall
treatmenttimeof22 days.Micewereirradiated usinga 20 x 13-mm
bilaterally fieldwhichincluded both
kidneys.Eachradiationdosewassplitintotwoimmediately consecutive withthemicerotated
halves, 180"
inbetween,toimprove doseuniformity.
X rays.Theirradiation
system hasbeendescribed previously(19,20).240kVpX rayswerefiltered with
1.0mmAl and0.25 mmCu,togivean HVL = 1.3mmCu. Dose ratewas 1.7Gymin-'.Blocksoffour
dosegroupswereeachgiven30 fractions ofthesameX-raydose,selected from therange0.2-1.6Gyper
fraction.
Fourgradedsingletop-updosesofd(4)-Beneutrons weregivensubsequently to eachblockof
dosegroupsto producea complete dose-response curveforeachsmallX-raydosestudied, accordingto
themethod ofJoinerandJohns (21).
Neutrons.Neutrons wereproduced from theGrayLaboratory vandeGraaffaccelerator bybombarding
a thickberyllium
targetwith4 MeV deuterons. Thisgaveneutrons withmeanenergy = 2.3 MeV,dose
meanlinealenergy = 65.6keV/pm, andhalf-value thickness- 3.0cmICRU muscle (22,23).Dosimetry

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 RENAL RESPONSE TO VERY SMALL DOSES 387

16 30 Neutron 16 30 X-ray (b) 30 X-ray

.215.1
Fractions Fractions 16 Fractions
1.45 0.8 2FN
E 12 .25 2FN 12 2FN 12
61.15 0.4
T1.6 1.3 1 06

a") 8 8 2 8-

4- 4 4-

-0 I 0 0
002 . 4 6 8 02. . . 4. . 6. 8 . 0. 2 4 6 8

Top-UpDose (Gy)
FinalNeutron
FIG.1.Residualactivityof5Cr-EDTAmeasured inplasmasamples1 h after plotted
injection, against
thefinaltop-updoseofd(4)-Beneutrons.
Eachdoseresponse curveis fora 30 smallfraction
"priming"
treatment,withdoseperfractionshown,followedbya neutrontop-up.The dose-response curveto the
farright
ofeachpanel(marked 2FN) showsa reference toa largedoseofneutrons
response splitintotwo
i.e.,zeropriming
equalfractions, treatment.
Errorsarestandard
errorofthemean.

wasaccording totheEuropeanProtocol (24). Dosesarequotedas neutron dosealoneinagreement with

ourpractice (21,25).They contamination
previously was12.2%ofthetotalneutron plusy dose.Neutron
doseratewas0.5 Gymin-'.Neutrons weregiveneither as twolargefractions
or as 30 smallfractions in
thedoserange0.05-0.25Gyperfraction followedbya largesingleneutron top-updoseaccording tothe
sameschedule usedwiththesmallX-rayfractions.
Assays.Threeassaysofrenaldamagewereused:clearance of"Cr-EDTA,urineoutput, andhematocrit
reduction as described
previously (19,20, 25, 26). Micewereassayedevery4 to 5 weeksfrom16to 50
weekspostirradiation.
Isotopeclearance."5Cr-EDTA was usedto assessrenalclearancebymeasuring concentration in the
blood1 h afteran intraperitoneal
injectionofI10 Ci (i 200jg EDTA) givenas 0.1 mlofa solution with
activity100MCi/ml. A single70 ,l bloodsamplewastakenfromtheorbitalsinusandcentrifuged for2
minat 12,000g, andplasmasamples werethencountedforresidual 7 activity.
Results areexpressed as the
percentage ofinjected permilliliter
activity ofplasmaat 1h after injection.
Hematocrit. reduction
Progressive in hematocrit is a consequence ofrenalirradiation (26) andis dose
dependent. Hematocrit wasmeasured aftercentrifugation in thebloodsampletakenduring theisotope
clearanceassay.
Urineoutput. Urinationfrequency, expressed inurination eventsper24-hperiod, isa convenient mea-
sureoftheurineoutput, whichincreases withtimein a dose-dependent mannerafterrenalirradiation
(19). Micewerehousedin individual wire-bottomed cagesfor24-hperiods, and urination eventswere
recorded ona paperrollmoving at 15cmh-' belowthebottom ofthecages.

RESULTS

In agreement
withourpracticepreviously,wechoseinitially toanalyzethedataat
thetimewhenmaximum and
resolution differentiation ofthedose-response curves
wereobtainedforeachassay(20,21, 25,27). Thisprocedure isjustified
sinceithas
beendemonstratedthatthetimeofassayhas no significant influenceon eitherthe
shapeoftheunderlyingdose-responserelationship (as determined bythea/3 ratio)
or RBE valuesmeasuredforneutrons (20, 21, 25). Bestresolution in theseexperi-
mentswas obtainedat 29 weekspostirradiation,and Figs. 1, 2, and 3 showthedata
obtainedwiththethreeassays.Smoothdose-responsecurveswerefittedto thedata
pointswithleast-squaresregression,
usinga generalizedlogitfunction.These curves

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388 JOINERAND JOHNS

45
30 Neutron (a)
45
Fractions (a) 45 30 X-ray
45 Fractions
(b)
(b) 45
30 X-ray
45 Fractions (c)

0a
C 35 -35- 35 -

-25 - 25 - 25-

1.6 1.3 1 0.6 0.2 2FN 1.45 1.15 0.8 0.4 2FN
.25 .2 15 .1 .05 2FN

15 15 15
0 2 4 6 8 0 . 2. 4. .. 6. 8. 0. 2 4 6 8
FinalNeutron
Top-UpDose(Gy)
FIG.2. The hematocrit
measured The explanation
at 29 weekspostirradiation. ofthecurvesis as for
Fig. 1.

tothelinesonewoulddrawthrough
appearidentical thedata"byeye"andhavethe
of
advantage beingobjective fits.
Each dose-response curvein Figs.1,2, and 3 is madeup ofdosegroups(mean
+ SE ofsixmice) which received30 identicalinitial"priming" treatments ofeither
X raysor neutrons.
The doseperfraction given is shown next to each curve.These
dosegroupsthenreceivedgradedsingleneutron top-up dosesso thatfulldose-re-
sponsecurveswereobtainedas a function ofneutron top-updose.Thesecurvesare
lefttowardlowertop-updosesas theinitialdoseperfraction
shifted re-
getslarger,
an
flecting increasing(although subthreshold) effectof dosein each ofthe 30 small
fractions.
The curveon thefarrightofeachpanel(labeled2FN) in Figs.1,2, and 3
showstheresponsetoa totaldoseofneutrons alone(i.e.,noinitialpriming treatment)
givenin twofractionsseparatedby 1 week. This curveprovides a reference forzero
primingtreatmentagainstwhichtomeasure theleft-shiftoftheothercurvesforprim-
ingtreatmentplustop-up.Singleneutron doses(as usedforthetop-ups)werenot

30 Neutron 30 X-ray 30 X-ray

50 Fractions (a) 50 Fractions (b) 50 Fractions (c)
25 2 .15 .1 .05 2FN 1.6 1.3 1 0.60.2 2FN 1.45 1.15 0.8 0.4 2FN
40 40 40

30 30 30

= 20 20 20

10 10 10
-

S 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8

FinalNeutronTop-UpDose (Gy)
FIG.3. Theurination measured
frequency at 29 weekspostirradiation.
Theexplanation
ofthecurvesis
as forFig.1.

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 RENAL RESPONSE TO VERY SMALL DOSES 389

7 (a) 7 (b) 7 (C)

0 6 6 6
5 Z 5 5
-o
E 4 4 4
E
S4X-ray
fractions
33 3 3

S 2 * 2 2
1 1
1
1 5'Cr EDTA Retention Hematocrit UrinationFrequency

000
0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0

Dose per Fraction(Gy)

FIG.4. Equivalent remembered dose(seetext)plotted
againstdoseperfraction givenin the30 small
neutron (0). Eachpointshowsa meanvalue? standard
(0) orX-rayfractions errorofthemean.Lines
arefitsto thedatapointsbyleast-squares Solidline,linearfitto neutron
regression. data.Dashedline,
fittoX-raydataintherange1-1.6Gyperfraction
linear-quadratic (seetext).

usedinthereference
curveduetoa dose-limiting
guttolerance
forlargesingleirradia-
tions(21).

oftheSmallDose Fractions
Effect
Underlying
Thetechnique forderivingtheunderlying effectperfraction interms ofan equiva-
lentremembered dose(ERD) hasbeenexplained indetail(18).Joiner andJohns(21)
havealsodiscussed howtoimplement thismethodtoanalyzerenalfunction datain
an experimentaldesignidenticaltothatdescribed here.
TheERD istheleftward displacement ofeachofthedose-response curvesinFigs.
1,2, and 3 relative
to thereferencecurvefortwofractions ofneutrons alone(2FN)
andisa measureofthetotaleffect ofthe30 smalldosefractions inunitsofequivalent
neutron dose.Sincetheresponseofmousekidneyto d(4)-Beneutrons has an a/l3
ratioof --20 Gy,whichmeansan almostlinearunderlying neutronresponse, the
ERD is almostdirectly proportionaltothetotalunderlying effect(or-log[target cell
ofthesmalldosefractions.
survival])
Foreachsmalldoseperfraction inFigs.1,2,and3,theindividual ERD wasdeter-
minedforeachmousein thedose-response curvebyusingthefitted 2FN reference
curveto determine theequivalent neutron referencedoseat theparticular response
ofthatanimal.TheERD wasthencalculated bysubtracting the neutrontop-updose
forthatmousefromthisreference dose.No dependence ofERD on levelofeffect
wasfoundinthisstudyandso meanERD andstandard errorwerecalculatedforall
themiceineachdose-response curve.
Figure4 showsthemeanERD as a function ofX-rayorneutron doseperfraction.
The pattern ofresponseis verysimilarforall threeassaysalthough theurination
frequency assaygave somewhat more variable as
results, noted previously(21). The
dataforthesmallneutron fractionsclearlyfit
a linearrelationship withdose, andthe
lines
straight drawn on thediagrams weredetermined by least-squareslinear regres-

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390 JOINERAND JOHNS

TABLEI
intheLinear-Quadratic
ValuesoftheParameters Model

Notional Parameters
inthe
X rays RBE
Neutrons limiting "induced
repair"model
a/f a/fl at lowdoses
(Gy) (Gy) (aN/ax) g dc(Gy)
ERD vsdose/fraction
(Fig.4)a
EDTA clearance 2.81 ? 0.67 _b 13.2 1.1
(1.48-4.14) (11.1-15.3)
Hematocrit 1.70 + 0.36 _b 15.6 ? 1.5
(0.99-2.42) (12.6-18.6)
Urinationfrequency 4.9 ? 2.7 b 11.3 ? 1.4
(-0.4-10.2) (8.6-14.1)
AllavailableRBE data 2.17? 0.33 19.0 ? 6.7 14.3? 1.1 5.7? 4.3 0.19? 0.07
(Fig.7) (1.51-2.82) (5.56-32.4) (12.2-16.5) (-2.9-14.2) (0.04-0.34)

Note.ValuesderivedfromFigs.4 and7. Values? standard errorofthemean;95%confidence

limits
areshowninparentheses.
a Bestfits
toallthedatapoints.
b
Analysisnotpossible data.
sincedoserangeis4a/fl forneutron

sion.The intercept on theERD axisforeachassayis similarto thatobtainedpre-

viously (21). Thisdisplacement fromzerooccursbecausethetop-updosesweregiven
as singleneutron but
irradiations, the curvewasfora two-fraction
reference neutron
treatment. Thistherefore demonstrates thesmallbutfinite dose-sparing a single
as
2.3 MeV neutron doseis splitintotwofractionsand showsthatevenforthesehigh
LET neutrons itispossibletodetectsomerepairbetween twodoses.Thisresultindi-
catesthattheexperimental design wouldbe improved the
bygiving top-up dosesas
wellas thereference curveas twoneutron sincetheERD versusdosecurves
fractions,
(e.g.,Fig.4) wouldthengo through theorigin.In factthischangehasbeenimple-
mentedsuccessfully inmorerecent studies.
SinceERD is verynearlydirectly proportionalto underlyingeffect
(as definedby
-log[target cellsurvival]),thenFig.4 maybe thought ofas a setofinverted "cell
survival"curveswiththeoriginat theERD axisintercept. ThusLQ relationships
maybe fitted totheX-raydata,oftheform
(ERD - I) = W(d(a/fl)+ d2) (1)
whereI is theinterceptofthelinethroughtheneutron fractions
withtheERD axis,
Wis a scalingconstant,a/fl to
applies X rays,and d is thedose per The X-
fraction.
raydata in Fig.4 are notwellfittedbytheLQ relationship. Ifall pointsare used
in thefit,data points< 1 Gy perfraction tendto lie slightlyabovetheline,thus
demonstrating greater whilethose> 1 Gyperfraction
X-rayeffectiveness, lieslightly
below;i.e.,theresidualsarenotrandomly distributedwithdose.Theparameter val-
uesforthesefitsaregivenforcompleteness inTableI, although thepoordescription

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 RENAL RESPONSE TO VERY SMALL DOSES 391

ofthesedatabytheLQ modelshouldagainbe emphasized. Thisisoccurring

inspite
oftheveryrestricted The linespre-
dose rangecovered(0.2-1.6 Gy perfraction).
andabove.The
sentedin Fig.4 showtheLQ fittojustthedataat 1 Gyperfraction
in the
fitis nowgood therange1-1.6Gy,but discrepancy below1 Gyperfractionis
evenclearer,againinthedirection
ofincreasing
X-rayeffectiveness.

DISCUSSION

OtherMethodsofConstructing Curves
Dose-Response
Inthispaperwehavedescribed theanalysisoftherawdataat29 weekspostirradia-
tion,sincefortheseexperiments thiswasthelatesttime,giving themostpronounced
response, beforeitbecamenecessary tosacrificesignificantnumbers ofanimalsenter-
ingacuterenalfailure. Thusthebestresolution wasobtained, consistent withmaxi-
mizingthenumber ofanimalscontributing tothemeasurements.
Lebesqueetal. (28) showedthatforone particular setofdata,a "linear"rateof
expression of renal damage could be obtained by measuring theslopesofthelines
whenresponse, defined EDTA activity),
as log(% residual wasplotted againsttimefor
eachdosegroup.Theserateconstants couldalsobe usedtoconstruct dose-response
curves.Theanalysis ofisoeffectdosesderived from thesecurvesagreedgenerally with
theanalysis performed previously on thesame setof dataat a giventime afterirradia-
tion(20).
We appliedthismethodto ourdatabutwereunhappy withtheresults.We found
thattheexistence ofan additional quadraticcomponent (responseoctime2)inthese
response versustime relationships unpredictable noticedcasesofpositive,
was and
negative, and zero curvature on thelines.As pointedoutbyLebesqueetal. (28),the
presence or absence of these higherordertermsstrongly influenced thevalueob-
tainedforthelinear rate constant. Therefore,since our own setofdata wasnotchar-
acterized bya consistent response time
versus function, rejected approachin
we this
favorofusingtherawdata(Figs.1,2, and3).
A lessdetailedapplication ofthismethodwasappliedsubsequently byStewart et
al. (16) intheanalysis ofrenalresponse. Intheirapproach, unweighted second-degree
polynomials werefreely to thevaluesoflog(% residualactivity)
fitted versustime,
butrather thanusingthepolynomial coefficientsto generate dose-response curves,
thisprocedure wasusedonlyas a wayof"timeaveraging" theresults,byobtaining
interpolated response valuesreadofffrom thefittedcurvesata specific time.Theaim
ofthiswas to reducevariability in thedose-response curvessince fluctuationsin
function measuredat sequential testswouldbe smoothed out.We havetested this
methodon thepresent setofdata,and Fig.5 showsthedose-response curvescon-
structed frominterpolated valuesforthe clearanceassay,read fromthe time-
smoothedcurvesat 29 weeks.A comparisonof Fig. 5 (smootheddata) withFig. 1
(raw data) confirmsthe observationof Stewartet al. (16) thatthisproceduredoes
not changethepatternof response.For our own data, thedose-responsecurvesare
smoothedslightly,but overallno changein positionof thedose-responsecurvesor
reductionin theerrorson individualdose groupswas found.We therefore feltagain
to use theoriginaldose-responsecurves(Figs. 1, 2, and 3) for
thatit was preferable

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392 JOINERAND JOHNS

30 Neutron (a) 16 30 X-ray (b) 16 30 X-ray (C)

16 Fractions Fractions Fractions
2 .15 1 2FN
E 12 .25 .05 12- 1.6 1.3 1 0.6 2FN 12 - 1.45 0.8 2FN
1.15 0.4

8 8 8

- 4-
4 02

1
0 0 10 O
-- 2 4 6 8 0
45 2 ' 468 0 2 4 6 8
S0

FinalNeutron
Top-UpDose(Gy)
FIG.5. Datainterpolated
at29weeks,from
thesecond-order
polynomial
fitstotherelationship
between
log[residual
activity/ml
plasma]
andtime(seetext).
Theexplanation
ofthecurves
isas forFig.1.This
method
ofhandling
thedataproduces
almostidentical curvestothoseinFig.1.
dose-response

subsequent analysis,whichwouldthenremainas closelylinkedas possibleto the

originalmeasurements.
We havealsolookedat theinfluence ofassaytimeon theERD measurements in
Fig.4. Analysisat 41 weekspostirradiation
demonstrated thesamepattern ofERD
versusdose,although thevariabilitywasgreater
sincetherewerefewer miceavailable
foranalysisatthistime.Thelackofinfluence ofassaytimeon theresultsobtainedis
a consistentfindingin thissystemprovided thatthetreatmenthas beengivenin an
overalltimeof <1 month(16, 20, 21, 25). In summarizing thissectionwe would
thereforesuggestthatin thisparticularexperimentaldesign,theefficiencyofdata
wouldbeincreased
collection andtheerrorsreducedbyusingmore dosegroupsinthe
but
experiment testing only two orthreetimeswithin 25-35 weeks postirradiation.

DoseforX Rays
FittotheLQ ModelandEstimateofFlexure
Joineretal. (17) haveshownthata sensitive measureofX-rayeffectiveness atlow
dosesperfraction is therelativebiologicaleffectiveness
(RBE) betweenX raysand
highLET neutrons. TheRBE istheratioofX-raytoneutron doseperfraction needed
forthesameeffect. Iftheunderlying neutron response is linear,as in thiscase (Fig.
4),thentheRBE value(as itsreciprocal) reflectsexactlytherelative effectivenessper
grayofthatX-raydose.Relativeeffectiveness pergraymaybe defined as theslopeof
thechordjoiningtheoriginofan X-raycellsurvival curveto theX-raydosepoint
on thatcurve.An advantage in usingthisapproachis thatunknown artifactswhich
mightappearinthetop-uptechnique shouldbe similarforboththesmallX-raydose
fractions
and thesmallneutrondose fractions, and so shouldbe cancelledoutin
theRBE.
Since equivalentERD values implyan equivalenteffectiveness oftheinitialtreat-
ments(18), then RBE values were calculated fromFig. 4 as the ratio of X-rayto
neutrondose per fractionrequiredto give the same mean ERD. The RBE values
obtainedin thiswayare shownin Fig. 6. Each data pointwas derivedbytakingeach

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 RENAL RESPONSE TO VERY SMALL DOSES 393

14 (a) 14 (b) 14 (C)

12 12 12
10 10 10

8 ' 8 8
6 6 6
4 4 4
2 Cr EDTA Retention 2 Hematocrit 2 UrinationFrequency

0 ' 1 0
0'1 1 1 1.5 1
0 0.5 1.5 0 0.5 0 0.5 1.5

X-RayDose perFraction(Gy)
FIG. 6. RBE betweenX raysand neutrons
determined
fromthedata in Fig. 4. Pointsare means
? standard Thedataindicate
error. RBE below0.8 Gyperfraction,
decreasing reflecting
increasing
X-ray
as thedoseperfraction
effectiveness isreduced.Thelinesshowthebestfittoallavailabledata(seeFig.7)
usinga modelincorprating at decreasing
X-raysensitivity
increasing doseperfraction(seeDiscussion).
Thedottedlinesarethe95%confidence onthemean(expected)
limits valuesforthisfit.

X-raydosepointin Fig.4, calculating theequivalent neutron dosefromthebest-fit

linethrough theneutron data,anddividing theX-raydoseperfraction bythisvalue.
Thestandard error on eachmeanRBE value(Fig.6) wasderived usingFieller'stheo-
rem,fromtheSEM on eachmeanX-rayERD value(Fig.4) andtheerroron thefit
to theneutron datapoints.Alltheassaysshowa reduction in RBE belowabout0.8
Gy per fraction,although for urination frequency considered alone,thismaynot
be
actually significant.
Sincethesizeoftheseexperiments wasnecessarily tomanageable
restricted propor-
tions,we studied here only a restrictedX-ray dose range from 0.2 to 1.6 Gy per
fraction.However, tomeasure theflexuredose,i.e.,theX-raydoseperfraction below
whichno significant further increase intotaldosewouldoccurwithfractionation, or
morecompletely, toassessifandbelowwhatdosetheLQ modelfails,itis necessary
to putthesedataintothecontextoftherenaldose-fractionation relationship over
thecomplete range ofdoses. Again, thiscan best be doneinitiallybyconsidering the
RBE ofX raysrelative to highLET neutrons, forthreereasons(21). First,theRBE
versusdose relationship has beenfoundto be independent ofthelevelofeffect at
whichit is calculated,thusallowingdifferent experiments withnon-overlapping
ranges tobeintercompared.
ofeffect Second,allthreeassaysmaybeincluded without
theneedtoderiveequivalence relationshipsbetween thedifferentmeasures ofdam-
age.Third,thetop-updatamaybe compareddirectly withdatafromconventional
fullcoursefractionation, sinceRBE is a simpleratiocalculation inbothcases.
Figure7a summarizes allthe datacurrently availableon the RBE ofhighLET d(4)-
Be neutrons relativeto X rays,forrenaldamagein themouse.Thesedataaretaken
fromJoiner andJohns(21) andStewart etal. (25),withthepresent dataaddedas the
closedsymbols. Thereis excellent agreement acrossthewholedoserange,inspiteof
combining herethreeseparate setsofexperiments carriedoutover4 years, andthree
different The
assays. reduction in RBE below 0.8 Gyper fractionis seenhere clearly
as a featureofthewholeRBE versusdoserelationship and amountsto an increase

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394 JOINERAND JOHNS

14 (a) 200oo (b)

Coursell
Furl
12 Fractions Top-Up
""1
40 2-
10 L 0 i
S60i
>' -, 40o

0 101
0.1 .2 .4 6 1 2 4 6 10 20 20 10 6 4 2 1 .4 .2 0.1

X-RayDose perFraction
(Gy)
FIG.7. Allavailabledatafrom comparisonsbetween240kVpX raysandd(4)-Beneutrons inthemouse
kidney,plottedas (a) RBE between X raysandneutrons X-raydosestogive3%residual
or(b) isoeffective
40% hematocrit,
activity, or 15 urinationeventsperday,at 29 weekspostirradiation. Closedsymbols,
presentdata.Opensymbols, previousdatasummarized byJoiner andJohns(21). Squares,EDTA clear-
ance.Circles,hematocrit.Triangles,urinationfrequency.Thesolidlinesarethefittoall thedatausinga
modelincorporating increasing atverylowX-raydosesperfraction,
X-raysensitivity withthedottedlines
showing the95%confidence limitson themean(expected) valuesfromthisfit.Above1 Gyperfraction,
themodelrapidly approaches a simpleLQ fit,
as shownbythedashedline.

in X-rayeffectivenessbya factor of2.1 as thedoseperfraction is reducedfrom0.8

to0.2 Gy.
Analternative morecommonapproachtodiscussing theX-raydose-fractionation
responseis presented in Fig. 7b. ThisshowsthetotalX-raydose whichwouldbe
neededina fullcourseofmultiple equalfractions toachievea giveneffect,plotted as
a function ofdecreasingdoseperfraction. IftheLQ modelremained valid,thiscurve
wouldrisewithdecreasing slope,becomingflatat thelowestdoses(dashedline).
The effect levelchosenwas 3% residualactivity in theclearanceassayat 29 weeks
postirradiation,corresponding toa hematocrit of40%andurination frequency of15
events/day. Fortheexperiments includedin Fig.7, thisresponselevelcorresponds
toa meanneutron totaldoseof7.4 Gy,givenintwoequalfractions. Thedatapoints
in Fig. 7b werederivedfromFig. 7a at each X-raydose point,by usingtheRBE
valuetoobtainan equivalent neutron doseperfraction. Bycalculating thenumber of
fractions neededofthisneutron doseto reachthesameunderlying effectas 7.4 Gy
ofneutrons givenin twofractions, and assumingthatthesamenumberofX-ray
fractions wouldbe neededat thatX-raydoseperfraction, theX-raytotaldosewas
obtained.Fortop-upexperiments, thesetotalX-raydosescalculatedinthiswayare
mathematically tototalX-raydosesextrapolated
identical directlyfromtheneutron
top-updoses(see appendixofJoinerand Denekamp(29) forthiscalculation). The
closedsymbols inFig.7barethusestimated totaldosesofX raysgivenina fullcourse
ofdose fractions of0.2-1.6 Gy,whichwouldbe equivalent in overalleffect to the
combination of30 X-rayfractions ofthissizefollowed bya neutron top-updoseas
actually givenintheseexperiments. Forthesecalculations,weassumedthattheneu-

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 RENAL RESPONSE TO VERY SMALL DOSES 395

tron(andhencetop-up)response isdescribed bytheLQ model,andan a/flof19Gy

hasbeenused(TableI).
Bothapproaches inFig.7 suggest a deviation ofthedatafrom theLQ modelbelow
X-raydosesof 1 Gyperfraction, witha flexure doseformousekidneyin therange
0.8-1.0Gyperfraction. In theexperiments ofStewart etal. (16),up to 80 dosefrac-
tions(without top-up)wereusedto measurerenaldamageatdosesdownto0.75 Gy
perfraction. Thisstudyshoweda deviation fromtheLQ model,alsointhedirection
ofincreasedradiation effectiveness, below2 Gy although somefractionation effect
wasstillseen.Theseresults agreed with previous data ofStewart et al. (20) using up
to 64 fractions (dose per fraction - 1 as
Gy) reanalyzed by Lebesque et al. (28).
Stewart etal. (16) havetherefore suggested a higher flexure doseof 1-2 Gyperfrac-
tion.However, whilenotexcluding a realreduction in repaircapacityat decreasing
dosesperfraction below2 Gy,theypartially explaintheseresults byincomplete repair
betweenthelowestdose fractions, sinceto achievea constantoveralltimeforall
treatment schedules and limitedbyirradiation withintheworking day,a 5-hinter-
fractioninterval wasusedforthe80,64, 60,and40 fraction treatments (0.75-2 Gy
perfraction) compared with>I1dayforthelowerfraction numbers (2.5-12.5Gyper
fraction).Withthetop-uptechnique, we wereable to use a minimum interval be-
tweenfractions 7
of h, even for thelowest doses perfraction, and this more complete
repairmayexplainourlowerestimate offlexure dose.
Theinterfraction interval maybe important sinceRojasandJoiner (GrayLabora-
toryAnnualReport,1986)havefounda repairhalf-time (T11/2)of 2.1 h for2-Gy
fractionsinthemousekidney, although Stewart etal. (16) pointoutthata T11/2 > 3.3
h wouldbe neededtoexplaintheirdatafully usingtheLQ model.
In theratspinalcord,Angetal. (14) also foundthatas thedoseperfraction was
reducedbelow2 Gy,theincreasein X-raytotaldosewasmuchlessthanpredicted
fromtheLQ modelas fitted to higher dosesperfraction, wherethea/l ratiowas 1.7
Gy,a lowvalueas inthekidney. Eventhough theseauthors usedinterfraction inter-
valsofonly4 h in someofthetreatments withthelowestdoses(1.3-1.8 Gy per
fraction),as inthekidney, theseresults couldnotbe adequately explained byincom-
pleterepairusingknownvaluesofrepairhalftime.Angetal. (15) alsofounda 2 Gy
perfraction limitbelowwhichno further additional increasein totaldosewasseen
in mouselip mucosa,although theseauthorspointoutthattheseresults contradict
thoseformouseskin(17,30),wherea measurable fractionation effect, inagreement
withtheLQ model,wasseendownto 1 Gyperfraction. However, Joiner etal. (17)
also showedthattheLQ modeldid breakdownin skinbelowI Gy perfraction,
withthesuggestion thatskintolerance actuallydecreasedwithdecreasing doseper
fraction,i.e.,thatX-rayeffectiveness pergrayincreased as thedoseperfraction was
reducedfrom1 to 0.1 Gy,in directagreement withthekidneydata described in
thispaper.
In contrast to thesedata,Parkinsand Fowler(31) haveshownthatin themouse
the
lung, LQ model describes theX-raydose-fractionation relationship atleastdown
to 0.6 Gy and possiblyrightdownto 0.15 Gy perfraction, witha/fl ~ 3 Gy. It
therefore appearsthatforseveraltissuesthereis a moveawayfromthelinear-qua-
draticprediction towardincreasing X-rayeffectiveness below1-2 Gy perfraction,
butthatthisis nota universal phenomenon.

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396 JOINERAND JOHNS

ModelingtheData
To explaintheapparent extrasensitivityofmouseskintoX raysatverylowdoses
perfraction, we had speculatedpreviously (17) thatsensitive phasesof thecycle
wouldbe continuously replenished withcellsbycellcycleprogression andredistribu-
tionbetween low-dosefractions. As thedoseperfraction wasincreased aboveabout
0.8 Gy,thisreplenishment wouldbe gradually prevented byradiation-induced cell-
cycleblockingand thustheoverallresponsewouldthenbe determined moreand
morebycellsin resistant phases,thesensitive cellshavingbeenkilledinthefirst few
fractions. However,in kidney, thelabelingindexis knownto be low (~0.3%) as
confirmed recentlybyWilsonetal. (32) usingbromodeoxyuridine incorporation and
flowcytometry. Thereisevidence(G. D. Wilson,personalcommunication) thatthis
resultsfromnormalcellcycletimes(-- 1 day)butwithveryfewcellsin cycle.This
makesitdoubtful thatcellkinetics canadequately explaintheincrease inX-raysensi-
tivitybelow0.8 Gyperfraction.
Ifincomplete repairbetween fractions weretheexplanation, thensincewekeptthe
numberoffractions andtheinterfraction intervalconstant forall dosesperfraction,
thiswouldimplythattherepairratewas decreasing (increasing T1/2)rapidly with
decreasing dose perfraction. To explaintheresultsit wouldrequireeffective T1/2
valuesofabout2 h at 1.0Gy,3 h at 0.8 Gy,6 h at 0.6 Gy,17h at 0.4 Gy,and200 h
at 0.2 Gyperfraction. Ifthisincreasein T1/2wasduetorepairsaturation, thismight
imply much less of
availability repair mechanisms at thelower doses.We therefore
that
postulated increasing be
repaircapacitymight gradually induced as the doseper
fraction is increased from0 to0.8 Gy.Inducedrepairofradiation damage been
has
reported in human lymphocytes as a result
of to X
preexposure rays(33),butwith
dosesof0.01 Gywhichareverymuchsmallerthanusedin thepresent work.Addi-
tionally, to date this effecthas not been in
reported any other mammalian tissues.
However,it is worthpursuing sucha tenuousargument sinceitturnsoutthatthe
simplest modelthatcan be constructed fromthisreasoning fitsthesekidneydata
well,as shown below.
Letcellsurvival orunderlying effect(-log&SF)be described bytheLQ equation
-loge(SF) = ad + Od2. (2)
Let a decreasewithincreasing doseperfraction (dueto inducedrepair?)according
totheequation
a = ao(l + g.exp(-d/dc)). (3)
For zeroX-raydoseperfraction (d = 0), a = ao(1 + g) and henceg is a factorof
"extra"sensitivity, compared with thehighdose response(d > de) wherea = a0.
dcis a constant,i.e., a "criticaldose forrepairstimulation" lyingsomewhere in the
range 0-0.8 Gy. We chose to varya in thisfashionrather than 0, sincetheextreme
changesineffect (i.e.,-logeSF) wererequired atverylowdoseswherethead termin
Eq. (2) dominates over 3d2.However,it is also worthnotingat thispointthatthe
valueofa, i.e.,theinitialslopeon a survival curve,andthevalueof/ in Eq. (2) are
bothstrongly dependent on theproportion ofinitiallesionsallowedtorepair(9, 10).
Equation(3) wasincorporated intotheequationrelating RBE to X-raydoseper
fraction (21) and fitted to thedataofFig.7a usingweighted nonlinear least-squares

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 RENAL RESPONSE TO VERY SMALL DOSES 397

regression. theexpected
Alternatively relationship between X-raytotaldoseanddose
perfraction,basedontheLQ model(Eq. (2)),maybemodified withEq. (3) andfitted
to Fig.7b to givethesameresult.The solidlinesin Fig.7 showthegoodfitto the
datawiththissimplemodel,withparameter valuesinTableI. Thislineisalsorepro-
ducedin Fig.6. The unmodified LQ model is shownas a dashedlinein Fig.7; this
joinsthe fit
from the"inducedrepair"model (solidline)above 1.3Gyperfraction
and thusthevalueofca/1fromthismodelgivesthebestestimate ofthisparameter
forX-raydosesperfraction > 1.3Gy.The valueofa/03obtainedin thisway(Table
I) was2.2 Gy(95%CL 1.5-2.8Gy)andis slightly lowerthanthevalueof3.0 quoted
fromouranalysisofrenalRBE datapreviously (21), i.e.,justtheopensymbolsin
but
Fig.7, agrees wellwiththevalueof2.3 Gy from thelatestindependent study(16).
CONCLUSIONS

Theresults presentedhereforrenaldamageinthemousesupport theproposition

thattheLQ modeldescribes wellthedose-fractionation ina widevariety
relationship
ofnormaltissues, downto X-raydosesof2 Gyperfraction. In therange1-2Gyper
data fromtheliterature
fraction, castsomedoubton theapplicability of theLQ
model.Eachtissueshouldbe considered butthedatainthispapersupport
separately,
theuse oftheLQ modelforthemousekidneyin thisdoserange,provided thatthe
timebetween exceeds7 h. Below1 Gyperfraction
fractions wehaveshownherethat
theLQ modelfailsdrasticallyin kidney, in agreement
withourprevious in
findings
increaseinX-rayeffectiveness
skin(17), witha substantial as thedoseperfractionis
reducedbelow0.8 Gy.

ACKNOWLEDGMENTS
Thisworkwassupported bytheCancerResearch
entirely Wearealsograteful
Campaign. toDr.Howard
Thamesforsuggestions themodeling
regarding ofthesedata.

RECEIVED:September18, 1987; REVISED:November6, 1987

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