MtlII<>cI' fot E'''",,''".Il,st ofCbemIcaJ IlIiu", Ii""",".od Non·h"""," B.... _ £0:<»,,,,,,,,Ed><od by V B V_. G. C Buol<t D G. HoeI.od 0 B. P<oWe 19S1 SCOPE

Implications for Risk Assessmentof Genotoxic and Non-genotoxicMechanisms in Carcinogenesis

J. E. Trosko and C. C. Chang

ABSTRAcr

The genelic, somalic and ps)"holopeal heallh of human beings depends on ahier.",hy of horneosUolic .daphve mech.ni$Jl1S allh. mol«uLar, b,ochem,caLcellular .nd physiological level., Pos.ible ways are ....mme<l by ....hich chem,_Clny induced mul.Fn..... ")'IOIOX.:ily or gene modulalion miihl romribu~10

carei.oien..is, leraloi"""is and ..-prod""live dysfunc:l,on_ The ronceplS ofinilialion and promolion are introduced 10 d""'ribe lhe p,ocn. by ,,-hleh • Siniknormal "em cell rould be lran.formed 10 a 'lably allered ceiL ,,-hich i. lhendonally amp1i6ed 10 a .rilical ma.. of dysfunclionallis,ue.

B,oloi'CIl .ffect> of chem>eals cannOI be ao;urale!y predocle<! wilhoulkno"ini Ihe biolopeal ronleXl of lhe chemical inleraction "'ilh Ihe la'llel.Ahhou&h il is importanllO ch.raclenzc po~nl,albioloiOC.l effeclS ofchemic.ls.Ihe amounl of uneenainly buill into individual bioloii...1orpni.ms (includi"llienelic, developmental. nUlnuon.l.•nd physioloiocal fIClors) limn. our abilitylo .."mote,he prohabk harm lhal uposuresloch.mocal. may ClU.. 10 hum.nhealth.

1 Il>o,RODUcrION

In order '0 develop a mean. for quanlitalive e..imalion of risk in humans fromexposure 10 chemical•. 11 is necessary 10 pUI the problem inlo a dear conceplualframework. To ,hi..nd " .. ha.'e .ummarized our "'orkini h)"p<llhcsis in Fiiure l.Thi. conceplual ",h.me lmpl....leI lhree fundarnenlal biolopeal processcs­mUllgenesi.. C)~o'o,icilY and inl..cellula, rommunocauon-in lhe i.n..i. ofienelic disorders, coniOnilal d.fecl>. Clncer, reprod""u... d),.funclion Ind mherchronic di..a.... II is. 'herefore, ....mill 10 undersland Ihe basic mechanisms ofIh~ prco::esses.

II ~hould be obVIOUS from lhe 0""'1 lhal lhi. will nol be an ea,y IUk since

'"

182 M~'Jwds jor Ulim,wng RISk of Ch",ni,ol Inju,y

'0."""" TO

Fiprr 1 It. !><un,"c "'."",.. rot cla..,:r)'lllJ c!l<ml<al. on til< \>0... of t"reebiolOJiuI cndpo; n", mU'"Fn ieity. <)'Ioto»clty and inhibi ,00" of io'<=lIula,commung'ooo. Tbt ."0.... bo'....... til< boo\oJocal cndpo,o" ""'"" t!lo'cb<micall con lui... mulllpl< biolop:ol off"''' io th< direction indie.,ed. Fo'exompl<. mU"'J<II' COlI kill «II••nd ,II< <ldtb of 0<11. con cou.. til<modula'i"" of Jel'" e.pr6liOll ill SOaK "",..i.mJ o<lls. [n addi'ion...."'"cbtnticals "'bocb <all mblbit mt.rcellular c""""."ut"'" at fIotl<)10"'''C""..Is., CO<IId.•t "'Jher conceo,...."oo•. k,1I coli•. pcmlbly also lndoci0l sorn<cbromosomal mutotoo",

(1) nol.lI mUtagens ac! in tb. sam< way (fo, .>ample. g.ne m.U1tiOllS "mllSchromosomal mUlatioos: mUlagellS "'hich damall' DNA "e!'Sus th""" "h"bmodula" tM fid<1lty of rrpall and r~pbca"on of DNA):

(2) ch.mical. can be <yloto.ic for ' ..nous reaSOns (for m"ance. desltoymemh....ne function: "'hibit impoflanl,ozym< functions; damall' Of motaleDNA. etc.);

(3) inhIbition of in'et«llula' communIcation by chemicals is probably' a,compl•• as mutall.ne,j, aod cell killing: .nd

(4) lhesc prOC('Sst$ arc not mulually eadu'i.... i,e. chemicals "'h"h causemUlalion, can also be C)'loto.ic "'hicb could lead 10 inhibition ofSOme formsof inle«<lIula' communicahon.

In summ.ry..... ,,'ill d.fin. chemicals,. hieh all.. lhe genetic informalion ofcell. (at lhe gene and chromosomal k"e1) as mUlagens.•,he",.. chemicals,. hichaller Ih. expfCSSlon of lhe genetic information ",11 be ";''''ed .. non-mUlag.nlC("yloto..os and InhIbitors of ,nt.«<lIularcommunlCa"on). $11""..... are a....re ofIhe lendency 10 use the lerms 'aenoloaic' and ·.pig.netic· in a ."Cry loose mann",(wllholll a d.ar d.finllion or inconsi".nlly). "'e ,,'ill consider 'genotoaie' to becqui"alentlo Ih. br""desl meanini of 'mutagenic' (i ......fcmnglo mutations atbelh lhe g.n. and chromosomal Ie,·els). and ·.pigenetie· to ref.. 10 themodulation ofg.n•••p......lon at Ih.,ranscripllonal ro posl_lranslallonallC\,.l.Wily. "hen these lenns arc used 10 describe ch.micals as i.nOlO." orepijenelic. il is not assumed that il"'ould preclude the.. chem""'ls from bav,nll

th. oppo$'" aellon under d,tT.... nt eil'C\lmslanc.., The l.rm. iI.nO\Qxie and,plg.nelic ref.r 10 .pecifie mechani.m. of aWon, Ch.mleal. aln .heit ditT....ntbiological eon>C<!u.n=. d.pendmg on eircumslan= (for .....mpl•. diff.r.mmelaboli,m In .pecies. lissues. d,,'.lopmenlal stall"' or eonct;nlralions u...:l).

2 :\IULTISTAGE :"ATURE or CARCINOGENESIS ANDITS IMPLICATIONS

W. wIllI')' 10 i\lU$lr~t. m lh. followIng di\euS\ion SOlO. of lh. faclors thoughllomftu.nc<' earc'nog..' ..... 'n human bein", Hov.·O\'.r. because of ,h.l,mllalion. oflhi. repon. wc win ha". to gen.rallZe some of .. hat "'e f~1 a... lhe imponampoinlS, In-d.plh analy.i, of Ih. follo..ing di",u..ion can be found In olher...pons (Trosko and Chanil. 1978a,b, 1979. 1980, 1981, 1983a,b),

It i. now ,,·id.nl lhal eareino~nesl'" a eompkx prOct:SS m'ol"ma the clonal.xpansion of a .inill. allered ct:ll (Fialkow. 1974; Cairn,. 1975: No..,.lk 1976:Bayhn or ~/.. 1978). Th. eonct:ptS of mllll"ion and p,omOllon ha.'. beenposlulaled to .xplain expert/n.ntal carcinogene.i, .tudi.. in animal.(Ber<nblum. 1941, Rous and Ktdd. 1941, MOll,am. 1944: Bou'....n. 1964) and.pid.miological obse,.,.-ations of human carcinogen.." (Amlllage and Doll.19~: Hakama. 1971, Wcber and Heck.r. 1978: Redd} 01 al. 1978: Dom.nof,1979; Moolp"ka"l ~I.• 1980~

The inilialion prOct:ss appears 10 in'ol".th. perman.nl all.ration of ,peclficgen., 10 a coU Oflh••xposed organISm. whIle promollon Sttm. to ,,>elude lhoscp,Oct:>Se$ whleh can allow lh. sele<:,i"e and clonal .xpansion of Ih. inilialedcell (poll.r. 1980: Trosko and Chana. 1980), a.arly. lhe sp.,,,fic molecularmechan"ms fore"her th. Inlhahon 0' lh. p,omouon phase ofeareinOilcn.,,, arenOl known. allhough lhe characteri'tics of inilialo" and promoters appear 10 bed""r>el (Barrell and SI.skio. 1980), Chemiea.l mUlagen•. ofCOUrse. by d.finll,oncan lnd""" permanenlalleralion, of~nelic informalion. a propeny of inilialOrs.and lhey. ofcourse. hay. been .hown in most cases to be carcinoaen. (Ames n111, 1973: Trosko and Chanil. 1979). On Ihe other hand. many chemiea.ls... hichha". 001 been .hown 10 be mUlas.noc under b,olol'cal cond,llons. ean Inftuenc.lhe promollon phase of earcmOil.neslS (T rosko n aL 1982b), These promolingeh.micals appear 10 enhance. all.asl. lh. sel""I"" h)"perplal'~ ofthe lmhaled ""II(Trosko and Chang. 1980) and 10 IOflu.nce. amona olher lhin". lhe exp..."ionof ilenelic information, .itber 1»' g.ne modulauon lhrouiih eOZ}'me md""\lon orby .000m. aet"'~lion (Trosko and Chani. 1983a). On. commonly, but by nomean. universally. accepled hypolheSl< "Ihal ,nillinors \e<m 10 be mUlaaens(FnOloxin.) ..hile promoters appe~r10 be •eplil.nelic· (non·s.noloxin,)(Troskoand Chang, 1980), All.mali,'e mechanism' by .. hich In,\lalors and promolcrsacl invol". inlllalors aI ind"""" of ilenelic "an,posilion, (Fahmy and Fahmy.1980; Cairns. 1981: R.nan. 1981) and promolers a' inducers of recombinationm""hamsm$ (K 'nsclla and Radman. 1978: Nasa""'a and Linle. 1979: Gennl ~,

IS4 M~lhods for £.ni""'''''fJ Rut of Ch~mjr(J1 InJu,y

al., 1980: Va~ha"s~y, 1981). chromosomal aberrallom (Ernenl and Ccnllh.1981) and aneuploidy (Parry' tf al.. 1981). For the sake of anall~'" 'he>ealtemati"e postulaled m«haOlsms and their e,poenmen'al basi' ,,'ill not beextenoi"ely discu..ed, 'ince they have been re,·,O""ed pre"iou,ly (Trosko andChang. 1983b).

It should be understood. howe"er, 'ha' nOt all <ell, exposed '0 initiators are'initia'ed', An initiated cell is a cell ",'hich ha' a specilk alteralion in ilS genomewhich hasconverted" from a normalcell'o a 'p=hgnan" ctlL In addi'ion, the'erm, ',ni,ia'or' and 'promo'e" refer 10 lhe mechani'ms of action of chemicalsunder ,pecllk cond",ons of Uloe. and nol 10 m'nnslC propoer1le< of'he chemlC".!. Inolher ""ords, a mutagenic chemical mighl be an mi'iator ,,'hen apphed in non·cy10'o,ic doses. whcrea, the same du:mlCal. g,,'en to an mltlated an'mal at highdo..-s. mliht 'promo'. the pf'e\iously iniliated cell 10 become a tumour by itscy10'o,ic ac'ion and the compensa'ory hrperplasia i' would indoce

The h)'polhe<" lhal carci""llenesi, can be expla,ned by Ihe initialion andpromotion concept>, If accepled, impli.. tha' the Inilialion and promotionproc....-s could be prO\'enled or suppre,sed (an'i·iniHalion, an'i-promolion) orenhanced (Trosto and Chang. 1978a, 1980). In addItion, sinct it is well kno"'nthat th.re are man) '-anables "hich can alter ,he biological .xpresslon of acarcinog.n in ,he ho,t (for ....mple. genet.. factors (Tro,ko and Chang, 1978b;Trosto tf af.. 1983), numtional statuS of the organism (Newell and Ellison,1981). synergIsm, Or antagon,sms with olhrt drugs (Trosko and Chang. 1983a.).psychosocial factors (R,Ie), \981)). II seems to us 'ha' tradi'ional concepu of'threSholds' and 'camnog.n,' (Trosko and Chang. 1983a) are not onlymisleading. they are. ,n elfect, usele.. since the)' do not connote the specificmechanisms or Imply' the complex mterplay of 'hese factors. Based on thea..umpllon that ,n,llallon and promotion conceplS do ade<juatel)' explain thecarcinoll"nic process, Ihere mu't be genetic. de"elopmental, phYSIological,nutri'ional and emotional factors ",'hich can 'pred"posc' Indi,'idual. to eithe' oroolh of these processe' (as well U Ihe anti·processes). For exampl•. ifmulag.nesi, i. a,l.a" one oflh. mechanisms for inilialion ofprecancerou. cells,then genetIC faCtor>. such ll$lhose '" hich .nhance ,h. f""'lucncy ofDNA damageor th. erro'-proneness of DNA repair or replication, could enhance Ihe ri'kassociated with ini'iators. X.roderma p,gm.nlo,um. Bloom', syndrome andalb'nos miiht 61 into lhat calegory' (Trosko and Chang, 1983a; Trosko rl aJ"1983). In addl1lon. ChemICals which might "'th...nhance or reduce anolheragent', potrnllal to damage DNA (for e...mple. photosensitIZe" or drugmetabolizing chemical') could either enhance or reduce the innia'ion .ndmutation frequcncy (Trosko and Chang. 1981).

By direcl implication ofthi. hypothesis. inllialion or mUlation ofspecllk genesm a cell" a nece""ry, but not sufficient. Slep in carcinogenesis (Poner, 1980;Trosko and ChanJ- 1980), Clurly. th. pathogen..i, of Cancer Involv.. lhe"'olu'ion of phenOlypes '" hich il\'< a precancerous celilhe ability to escape the

ttjulalory mechanism. of th. body to di,'id. and d,fferon,,,,,e properly and toha,.. unconlfolled in,,,s",'e properl'" (Fould•. 1975. Nicolson, 1979; Post. andFidl.r. 1980: Nicolson and CU'lead. 1982). If th. in"i..0<1 «II OC"u.... ,n anorganism "hose g.n.'ic, d""dopmenta1. nutrillonal or ph}'siological "alefawur. Or .upp....... Ih. donal e.pan"on of that «11. a tumou, "'ill .nherappea' or not

We ha,e d,><:u,..,j .lse,,-he.. that,nit;at;on probably """u.... ;n e",ry organi.m(",pomaneou,' 0' 'induttd'). and Ihal tho rat...hmning ".p i. probably lhepromolion phase (Tro.ko and Chang, 1983a), Thus. 11 's ,mperahw 'ha' w.unde"and 'he mechanism' and facIo.... ,,-hich can .ither .nhance Of ,uppress lhodonal ••pan,ion and ....olu"on of a .,ngle \n,,,..«1 ~.11 to a frank in,.."".'umour. The "ery fact that a ,mal. ,n;tiat«l cell can ",,,,,,,n 'quiesc.nt" for longperiod. of lime and that many g.n'lic (Trosko u at.. 1983), nu'ri,ional (Cruse.1(1/. 1978). ph).ical (Arg) ,i. and Siaga. 1981), ,ncludin& fo""gn bod,.. (Ryan .,at.. 1981), d.-,.Iopm."tat (Goemlcr and Loehrke, 1976), phy.iological (Yag.,and Yag... 1980) or ••og.nou. faclors (Hecker" a/.. 1982) can promote tho..in;lial«l cell' ><emS '0 undermine th. nOlion that an 'immune surv.illance's}'''em can ehminale many potenlial ,umour cell. (although it does nol impl}'Ihatlh. 'mmune .}'st.m play. no role in cancer de"elopmen,j

3 POTENTIAL ROLE OF INTERCELLULAR CO.\I.\IUNICATlO:>OIN TL:MOUR PRO:'>IOTlO:,\" TERATOGENESIS A:>oD

REPRODUCTIVE DYSFUNCTION

There are "arious mechani.ms by "h,~h «11. ~an Commum~at. (><e POller.1983). Th. function of an 'hose mechan;,m. i' 10 ...gulate. in multicellular anddiffer.nti.ted orgam.m•. the «11, of IlSSu"" to prohfcrale and d,ff.ren".,.(Loe"·enst.m. 1979; Piu., 1980; R...I<I "/,, 1980), F;gure 2 diagramma'-:allyillustra .... in broad general'e""s, 'wo fonn. of \n,.,cellular .ommunication.',',.0"" a distance via a molecular .ignal .uch as a hormon._ grow,h fa~'or orchalon•. and ,'ia gap junction, he''''..n physically conlilnou. cell.,

Ourly if w•. in prin~lpl•. accep' 'h. fundam.n,.1 role "hich mtercellularcommunication play. in th. d.licat. control that .I.m cells ha". on th.i,lerminally diff....nlialed dauah,.r cell' and m th. coord;nalion of diff.",nt,issues and of cells with,n a "SSU'. th.n ,,-. ~an lopcally ded~ 'ha' ehronicdisrupllon or di.ruption at a cri'ical .Iage of thi. C}'hemetic or homeo'laticfeedbaek ~on"ol $j'<!em will ~ause po1<n,ial adapti," dj'SfufIC"on. We and o,herin"estigaton; h3\" .peculated that ~hrontC inh,bit;on of intercellular communi­calion in init,al«lt'<sue ean. in pan. lead to tumou, promo'ion (Murray' andFitzgerald, 1979; Yolli., al" 1979, Polter. 1980). In add'hon. ",e ha,'. ,peculatedthat dlS",p"on of ,n'ercellular communication during critical period' ofde"c1opmenl might he on. of lb. causes of leratogenc\i. (Trosko <I<1i.. 198a), bperimenl.1 ,-',clerlC< in the form oflho ",.11 known ·ca",,~i.

186 M"MJs/<K £Jli"""~ Risk o/CMmk~llnftiry

lO'G .. ~>'O~r-l'V(OL(If<G .. S_T-OIsr""CE

SP£C'~«:"NON-Sl'EC"'C

CEll-CELL COIo1MUNICATION

FiJI'''' 2 ThI. d....ram ,Ul1<tra'" '''0 ,.....raJ fo...,. of ,nte,­cellular commu.ue.""n, On< 'n¥ol,.. ,I>< prod""""" and 'raru­",,""'n 0( -"par lI>Oiro.Ik> ""'"' • di....""" lhroullb .,."acdlular.pace '" • ,or"" ti>Slle. The OIlier ,n"ol'.. tl>< tron.fer of .......1'mok<:ules "a permeable in'ercellular jun<tlon. 1><",_ coupledcell.

te'alo,""",\,' <onnec'ion (Milk,. 1977; Nomura. 1977). and Ihe demonstrationthal man)' <hcm"'als "hleh ,nh,b'l ,n"""lIula' <ommun",at,on arc ."hertumou, p,omoters or 'e,alogen, or bolh (Fi,urc l), a,e <onslSlent "'ilh thathYPOlhes,s_

In addition. in ca.., ",he,e lhe malu,,"o" of .peclfi< cells. such a, spcrm cells.d<pendson an ,nhmate<ommun,,:al,on proc= ,,"h ne,ghbouring bu, d,rr.rt1ltcells (~r1oh and u,yd,g cdls) (Sha~ " al,. 1981). lhe inmfere.... wilh

"'" ...~.",,-­,'.._.-..

····1--1.l()() •

• l()l() •

• l()O •

••..,..,-F,Il''' l Th" <haJl"m ,ll~"ra ... a hH'otl>osi> I,nk,n, mU'a,......" ",II de.l.th andd"rup'ed ,n«",~lIul.o, e<>mm~nie.,jon '0 tbo ,nit..,i"" pro"",,;on of can:rnolC'\CSi. aodto ,...101<....... An <lIrly <or>«p<" Of f.t~., O'pooed to • ","ta,.n at • low <!os< <oUld<o""""..bly ha," • critlc.1 ,."" mutated ,n • ,«", ""Ulead'o, to a <on,.n"o] d<f«t, A'hlah. Ojo~oto.u< d..... of a muta,.", fetal 'oxieity ",,,uld bo <llJ'<Cted, hl'O'O" to """,.,.,,<>lO'ie, bo' Ojo'!OlO'''' cbomals could alo" Iud to~,talWecu '" fetal to.""t)'Chem"",I, "'hICh onh,b" ,n«..,.Uul., <omm~nie"""" ."uld d",upt re,ula""n ofprolof,..'",n and d,lf,,,nuatlO1l oft....... ,f I'veo at • ental penod of doe,-.Iopmen', 'Th<."n""l""""'" ..'ould bo ..,bor con,."ital Wecu '" embl)'<>- '" f<to'o"",,'y, 'Th< ..mechemical. aod ""00' of cbornieol, can 10ft...""" carelnol'''''''' 0' ,"bo, compl.te or'it><omple1e' caron"",o'

in'ercellula, communica'ion could block germ cell maluralion, One wouldpredict that chemial. "'hich inlerfe" ...l1h this spee,fic fonn of inlercellularcommuwcauon could lead 10 rq>roduetive dysfur><:tion.

Dearl)'. even if inte'cellular communocalion plal'S Iiule or no direct .01< ,n the

188 MrtAodsf()l" Eslu.wllltf/ Rosie ofCAem.callt'j~,y

tumOUr promot;on process. lhe faclthat lhere are chtmical' which disrupl thisimportant b,ological proc= at concenlration, Ibal do nol killlht cells. I~ad, u,ro 'pttulalr l!lcy can br harmful 10 human h~all.h

(I) iflh~ concenlralion is bigh enough 10 ,nh,brl in ..=lIul,,, communicalion:(2) ift!lc r~posun: 10 lh~ chemicals is regular and ch,onic.., as 10 pre'~nl n"rmal

h"m""".lalic c"nlm\ brlWttn Slem and daugh,.r cells, brl"'~~n r~gulalOry

and lar~ ,issu~ or br'Wttn funclioning cells of a giv~n li<slll': and(3) if presenl during crilical period, "f d"'rl,,pmrnl.

4 MUTAGENESIS, MUTAGE~S A~D MUTATlO~S

11 should be: apparen, lhal mUtIllions In lhe ge'm hne con",bu'~ to g~n~'ic

diseases. Allhough a re,'i~w oflh~ possibl~ rok of mulallons in somalic li..u~ 10o'her di<ea<e ,lales "ill nol be: made here. il i' highly likely lhall!lcy pial' somrrole ,n carcInogenesis (T rosko and Chang. 1978<). a'he,osderos" (Bendil1, 1977:Tro'ko and Chang. 1980). leralogen~";s (Wilson. 1977) and 'ag"ng' (Trosko andChang. 1976: Trosko and Hart. 1976). Con~u~nlll, il is essential1hal "-'understand lhe mechanism' of mutagenesis and lhe lechniqlll" 10 mea,ure,-anou.da,.... ofmu,alIon, and mUtllgens as well as Ihr spttific mura ,ions" hichmighl comribule 10 lhese disease,. Clearly. lhal kind ofcompn:hen,,"~re'·,...· "nol po..ibl~ her~. Suffic~ iliO sa)' lhal ".~ kno" ...ry l"lle abou, lhr m"le<:ulardelails of mUlagcnic me<:lianllmS ~spttlalll fo' mammalian and humanmUla""ns.

Firsl, ....~ mU'1 agree on a "'"rking definili"n ofa mUlagen. For "ur purposes amUlag~n is an agenl "h..h can, in prac"call~rmS. i'n:"~rsibly alter lhe origmalqualily "r quanlily of g~nClic inf"rmali"n. Thi' can be achi"-.d 1»' chemicals...h,ch mal' dIrectly 0' mdirectly damage DNA bases, DNA smonds. causeaddition. "r d~lelions. or lranslocation! or lran,positio", of chmmosomes"ucIure. Operalronally. 'e<:hniqlll'S used to mralU,r DNA damage can usuallyd~lect lh" cIa.. of mUlagens.

In addilion, ag~nl' which mighl nol damage DNA, bUl "hich could affectmembran~ cYloskel~ton SIT\iClure or arresl normal DNA synlhesis, could cauS<karyolyl"C changes (Huang~, al__ 1983). Furrhermore, Ihere ar¢ehemicals whIChdo not damag~ DNA 1M' H, bUI can alt~r Ihe DNA infotmllion conl~n{ byIn"racung ""h olher non·mutllgcns (COmulagenS, Nagao" (11.. 1979), byinhibiling DNA repair ~I1lYmes. 1»' chanaing lhe fid~lily' of polymerases(Wey'mou,h and L«b. 1978). by ah(nng nudeotide pool~ (Hopkin~ andGoodman, 1990; Anderson ~I (1/.. 1981). and 1»' aCli"alin8 deacli"aling pro­carcmogen~ carclno,en. (Langenbach.r a/.. 1978). ThIS does no, includ~ lhepossible 'ynerS'''.. ,n"cacuon betw«n ''''0 non'mulag~nic0' ...-.akly mula­genic a,~nl' or mUlag~nicaffectors (for example. m{eraClion of\'",ble hiltl "','hg.mrthoxypsoralrn. Bu,ge, and Simon•. 1979), Consequ~nlly. leslS 10 delect

mU'agens "'hich do not darnase the template DNA dlrtC,ly ",11 ha\'e '0 bede"elol""d. DNA damage and DNA repair a""Y' ,,'ill no' del""t agen.. affec'ina,p,ndle fibre. or aaent> "'h",h modula'e ,he fidel"y or normal DNA replicationThe~ are many k,nds of mu,at,ons at ,be sene and chro~ level•. The

me<hani.m, and kin.tic. of induetion of"anous land' ofmu'ation. a~ probably'qw,. dllfe~nt and ha\< not been workOll out ,n fine deurul for humanmutaa.tleSi•. Mo.t'e.t sy'tem•. ;~ ntro Or in 'iro.... hlOh a~ u>Cd to mea,ur.mu'ational change are 'indirect' m the ..= that they m.uure phenotypicchani'S, from "'hich "'e inf.r a chang. in the quali'y or quan'i'y of gen.,icinfonn.a'ion. Many of these 'e" system, ha\'e ..,..re Itmita'ion. (Tro.ko 01 oJ.,198ta.b). For .x.ample. u.ina a Nctenal ')51em '0 det""t pooslble hurnanmu'ag.ns. Can lead '0 SOme fal .. positi".s. if 'he chemical i. actinted to

mu'agenlC rorm by nitroreduetascs (enz)mes acti,·. in hacterial cen,) (Warren tI

0[,. 1982). AI..,.•n,'ironm.ntal. phy,iologkal and '''''hnICal factors related to thea""y can cause aro.. art.facts in the m.a'urement of mutat,ons. u.,ng ,he,-"o"e1)' of spe<:1fic phenotypIC chana" (,uch a, expression tim.. of mutation,.selceli,.. di...d,·antaac of mutant cell' in the population tested, 'metaboliccooperation', phcnocop}' induetion) iTrosko .1 ,,1.. 198Ia.b). T""hnlques '0'tudy th. ,'ariou. types of acne mutation, at the molc<:ular I."el are only no""being d."eloped, and the application of 'h... approaches to ...·,despr.adquan,itat,,'e and rap,d .. rttnlna " not yet poo,;ble

5 GENE MODULATION, GENE MODULATORS ANDINHIBITED INTERCELLULAR CO:\I:\IIJNICATEO""

The cells of a multicellular o'iani.m ,espond to chana" in ,he .n"ironment fornormal d"..lopm.nt. d,ffer.nulluon and funchon. How"'er. 'he chronIC and'critical 'ime' di.ruption of hom<>cos'atic mechani.m, can ha" a'herse con­<equen<:e' a. p,",'iously .UillestOll. Th. aeneral model ofa tran.membrane-aen.modulallon mechan'sm i. thouaJtt to allo.... specific cells to ,",pond '0 vanou•• n",onm.ntal slanals ,n .uch a manner 'ha' adap,i\< gen. 0' jlCne productfunClion comes into play, The role of int.ra:llularcnmmunica,ion in malntamlnahomocQSta,1C reaulauon of proltf.ration and diff'~n1ia,ion is beginmni to beundersll>od in some detail (Loc"..en".in, 1979). How.,..,. euCl detaIl< ofho....chemICals modulate spcciti<: gene or g.ne prodUCI wi"ity i. "ill underin"estiga'ion. It may he a"umOll that any chemical whICh im.rf.r......i'h 'hese,n'.rttllular communication-memb... ne acne ,riggers can ha"e detnmentalcon<equen=, del""ndina on circumstances (for eumpl•. su..ephblc tar!l"t ct'lis.critlCall""nod. of "'t<rfcfeflct'J,

Chenllcal.... hich can cause a potential rc"ers,bl. alteration In ,heexprc<,;on ofaenoli<: informatIon CQuid be conSIdered 'gene modulators' (epiaencti<: a!l"nlS).They might function at diff.rentle\..l. (DNA, or protein, or ct'llular) to causepheno'yp,c changes. Stem ct'lls, ...·hi<:h ha"e a 'choice' of prolif....tion or

19() Mellw<b for Ut!mhlinf/ Ri.!k 0/ CMm;"al f~jur!

dilTerentiatlon, are nonnally held in a slate of qu'e>CtllCe, Vanous m«:han'lrnS(for example. DNA meohylation. honnones. cyclic AMP. eakium. ~ll""",U

commumeauon) are IhoUllhl to be mvoh-ed in lhe '<:gulation of th. pr""",... ofpr01oferation and d,lT.renliation. Chemical. "hieh chanse or block normalr'llulalo')' processcs ",ithoul kliling Or mutallng lhe la'llel cell (for in'la~hormon.s or PBB,) can cau.. polential harm by all.nnll lhe panem. ofd.,;.lopm.nl and diffe",nuallon. as "".ll a. by d"rupllng normal organ funClion

Many chemical. in Ihi' Calegory have been "ho"'n to be r.lated to tumourpromouon (for .xample. hormones) and .nupromolion «)"10< AMP). Ofparlicular inleresl i' the demonst"'Iion thaI most 'U,peeled lumour promol....ha". lhe ablilly 10 block cell---«Il communicalion. A. pre,'iously hypothesIzed(Youi tI 01. 1979; Trosko and Chanll. 1980), under th. influ.""" of lhenonnaliling effect of surrounding and communlCatmg normal ~lIs. a sinileinitiated (or ll"n<tieally' defici.nt) ~n millhl not be abl. to ..pre.. its lumour (orolh" g<nehcally d.fici.nl) ph.nOll-pe. Th. inhibl1lon of inlercellular commum_<:ation (blockallc ofa <nlicalg.n. product) by~nam <hemical. could elimmat.lh" normall~mgelTectand con,'ert lhe InItIated (or genel>ea.lly detlei.n,) ~U inlOa lumour (or ph.nol ypicaU)' altored) cell. It should also be no,ed Ihat ,nhibnionof Inlercellular commun,cation can also be broughl oboul by c.lI removal(.url"l)'. ",oundlnll). mUUlge,,.,nduced cell kIlling. non_speeitle cytoto,i", andphy.ical blockag. (pLo$lic or metal ,urf""",,~

Th. technology to delec, ch.mreol. "'h,ch can Interfere wllh lhe vonou, fonnsof Intorttllula r commun.:at ion IS beIng de-'elopod (for .xampl•. elec"ocoupiln g,metabolic cooperalion, iS010pe or dye lran,fer (Murray and Fil~g.rald. 1979;Youi tI aI., 1919; t.:m.da " of., 1980; Kinsena, 1981; N.",bold and Amos. 1981;EnomOlO ,I 0/__ 1981. Trosko rI 01., 19810.b; Wilham••, 01.. 1981; Warren et 01..1982)). Th.re "'ill al"-..)"5 be inlrin'ic problem, in u,ing any jn "'''0 a,say to

predicl ill m'o hI"'''' ord", pr""",... (.uch as Speeles. 1llS1le Or developmentaldifferences), An .~ompl. of lhe problem of inl....pecie...trapolalion ho' been't'Cenlly analysed for hypo1op.daemre drug. In rodenl' and man (Co....n andGra.so.1981),

Howe,'or. if il tum, out lhol modulations ofgap jUncHOn, are cntreal,n pan oflhe membtane-tnllllered ,hif! in lhe ~11", phy'siologlCal and phenolyplC state,lhen il m''''l be lhal Ihe u.. of mosl ill ";"0 interttllular communication a Y'could detect all bUI lhe speeific receplor-<l.pend"nl membrane modulato .Although th. exaCI 'lr""tural and funcnonal hOmololl'cs of gap juocuon, m allmulticellular ,peei¢!; are nol kno"'n, il ",'ouk! nol be ,ufJIri,ing if al lea,t a highdegree of functional homology ha, been hIghly con""'ed lhrou'" e"olullon(Hertzberg. 1980; Nicholson tI 0/" 1981), Ther.fore, any agenl '" hreh <:an alTecllhe gap juncuon-<lcpendent ,mer~lIuLar communicalion ill ";/to could (dcpend­,ng on the ill 'Uri> di.lnbulion. concentralion and melaboli,m) be predicted toblock ,nlercellularcommun>ea.lion III .-in'. Th'" mlghl uplam" hy lhe <metaboliccooperallon' a.say. descnbed by You; tI 01. (1979), has detected 1no",n

promoters of lhe skin, bladder. hver. lung. cok," and bru'l ef several speci"".Oeull, in ,il'o model' 10 deteclpromoters have nel done a. well (for eumple.phenobarbnone or DDT are nOI mOuse skIn l""omOlm. bUI raIl",", promoters).

A polentlally new problem. from our perspeclive, i. that lhere are likely 10 hedifferenl k'nd' of tumour promolers. ,imilar to dlfferenl claS5e$ of In,llalors(direct indirttl ""hng: w,lh 'hort half-hfe. Ions half-life, elc,). In lhe ciusicstudie, on crolon oilpho,bol e.lers mouse skin lumour promOhon. 11 wouldappear 'ha' regular and long_term chronIC exposure at a 'Ihre,hold' Ie.'el of aprOmOler is needed in order for 'promOlion' to OCCUr (fIout"..l!. 1974: V.rma and801l1'''e1l. 1980). Th. m.taboli.m and .xcretion of Ihe chemICals "'hich indu.ee a""lenliall}' reversIble and adllpt,,'e res""nse (for .umple. m.mbrane modu_lation of ,on ftux and rcdox ""lenhaL di=lulion of pp junction.) wouldneces,ilate resular. chronic exposure 10 the chemical al a Ie"el which is highenough 10 rngger these even... Th" eaplanahon "",loS 10 fil Ih. ollse,,·alion. ofphorbol esler and phenobarbllone ptOmouon (chemlCals which a ... metabol1Ze<land .xcrel.d)o, of p,omolion by ...ceharin (a chemical .... hich I' not melabolized,but onl}' ...aches high enough concentralion. in ,pecific erpns such a. bladder).Ho......' ..r. Ihere " now' n;dence lhal SOme chemicals, for example lhe poly­bromlnaled biphenyl., are not ,ignificanlly metabolized (al lea" not lh.blol"i'CI-lly aclive congeners) nOr a ... they ucrered easIly. Once m Ihe body. lheyare stored in Ihe fal cells and an equilibrium is eslabli.hed in the body. thereby'OOlhlng' 'he body constantly. Mobilizalion of the ..ored bUI unmerabolizedPBBs during ph}'siololllCal shifts (for eumple, pregnancy) or dietary .hifts couldconcc,nbly cause long.term 'promo"on' from a "nllie exposure,

The pos"billly thallhere are 'Ihreshold" for indi,-idual promolers (floulwell,1974; Verma and BOul ......U. 1980)does nOI. ho...."(>'er. gIve uSlhe licence 10 exposeOUl"5<l\"es. ....,Ihngly, 10 many sublhresheld Ieo."'ls of I""0mole....ince have noSCienlllk evidence lhal lhere ....ould be no .ynerlJ'I;'; inleraellon of 1 '0 or moresublhreshold I..·el promOlers, If Ihe orpn dimibulion 0' concenlrallon ofdifferent ,ubthrc'hold promoters is d'fferenl, lben Ihere mlght no! be anyproblem, Ho...."'.'.., Ih" i, an area of research ,,'bere no informal ion " yela ..."lable. In adduion. conceplually. antlpromolers mIght also ameliorale theeffect of one or more promot.". If OUr thesis is correcl thalpromollon might belhe rate.limlilng "t!' of carcInogenesis. one can "'" thaI ln tbe 'real' ....·orld ofchemIcal. to whIch huma", arc exposed, the 'nel effect' mlgl" f!C'",r be predicledfrom information on ind..'idual substanceo,

6 CYTOTOXICITY AND CYTOTOXIl'i"S

lbe rol. ofcell dealh (or cell remonl) in lhe IUmOur promollon process has beenconSIdered by Fre; (1976) and Trosko and Chan, (1980). Parhal hepalc>:lomy,viralll Induced cell killing. cell dealh call$ed by mutagen. Or non-spec,fic

192 M~11uHiJ for £J'lmDl~ Rut oj CMmicallnjuTJ

C)10,oxm, (a~enl' deSlrolin~ membrane function, inhibitmg ~~Ial rnz~'meJ)

~n bnns abou' the ehmmation of m'erttltular communicauon ,,'ithou,mu,auns the DNA"r "'i,hou, aherinl sene expmsion dlf""tly,

Panial hepa,""lomy is a kno"" promoter (Pi,ot and Sirica. 19S0). Exposu,,,,'0 mu'asen. cartinOlens a' doses ,,'hicb do not cause .isnilkan' cell killinlu.nall)' b.a"e 10 be followed by promo,ers ('iocomple,e camnogens'). whereas a,cou«nu'ation. induc-inll celt killins. IhO)' are ·compl..ecartinogen, (Trasko andebanl. 19S1aj. CbemlCal...ucb as cbloroform and carbon te'raeblonde andpossibl)' TCDD, ,,'hicb are nol thoull!u to be mUlasen., do kill celts and arepromoters (Uebleke ~I of., 1977: Reuber, 1979; Potand and Gto"er. 1979; D,uGomel and Cawo. t980; P,tOI e' 01., 1980: ~"er and Neal, 1981; Kirkland tI

01" 1981), One polen,ial explana,io" is 'ha' tbese chemicals, by 'beir membrane·des'TllCu"e propeni"'. might 'nduce a Ca" to.ici'y (Kroner and Plunker, 1980;Farber. 1981; Cbenery 0' al.. 1981). Alcobol migh, be a tumour promoter. bykilling cell' ,'ia its membrane-allerinll rellulalion of Ca' - (Gold..ein and Ch'n.1981; Scb.anne" al.. 19S1j. In any case. cell death or ~o.'al by an)' mea""force. ,he .u"i.'inll ,Iem cell., ,ncluding a fev.' ini'ia'ed cell.. '0 proliferale.

7 CHE:\-t1CALS, CONTEXT AND CONSF.QUF.l"C£S

If il were a "mplo maller 10 ldenufy chemical. ,,'hich ei'her muta'e genes.modulate gene e.pres,iOll or kill cell., and 10 know how mu'allen",i.. senemodula"on Or cy,olOxicuy inftLltnct ,'anou. disease lIa'''. 'ben our abihly 10pTtdlC' Ibe consequences of human expo,ure to lhese chemICal. "'ould berela,ively ea.y, Alas. ,f .uch a d..ermmi,'ic relauon,hlp ex,sted be''''ecn 'p""ificchemicals and a Spec1t'>c b,olopcal disease stale. life would probably he a "eryIimiled. if no' precarious. Slate. Tbe 5<'ien'ific ,,'id~ 10 dale .bow. lhal thereare many faclors "hleh can ei'ber enbance, reduce or ameliora'e 'he po'en,ialeff""l of a 'p""i6c chemical in a 'pe<:,tic b1ololP<'al hOSL

We must cons,der the fact that a veal number of 'def~'meehan"m. mustbe o"ereome before a p"en chemical can aff""l a comple, b,ological en'i'y Orprocess. In addi'ion. once molecular or cellular larse" ba.'e been reached Ordamaged. 'bere are Ttdundant repalr and other compensa'ory meehanlSm'a.'ailable 10 protect 'he organi,m from damage, Tbe age a, which ,he animat orhuman orpn..m " exposed 10 ,be chemICal makes a Slgn,fican, difference in tbebiological ou\Come, Kilhnll a rev.' cri'ical ~II, ",'h a <yto,ox,n or ,nb,b",ng acri'ical intercell~lar communica'ion mechanism or mutalinll a soma,ic stem ~1I

for a gi....n organ d~ring ,be earl}' phases of embryonic d..-elopmen, may bebiololleally more de,'aSlating lhan ,be same exposure during lhe adull .lages orlife. In senerai. e.pen~ wllh agen...tlCh as X-rays and thalidom,de appliedduring cnl....1stages of early de."lopmenl bigblightl 'h.a' poinl

Exposure of a biolo,ical host to the same chemical but in different Clf­cumSlances. can apin brin, about Opposile effects, Exposure of anImals 10chemical ~arc\nOlen, after they were ..posed to agenls 'uch as phenobarbitoneor bUl)'laled hydroxytoluent S«TnS to re:due<: cancer ri'k (see re"ie.... by Tro.koand Ch.ang. 1978a. 1981),

On the other hand. the same chemical. Ii"en to lhe same an,mals after thO)'have hew exposed to a carcmOien enhaDCC the occurrence ofcancer (see revi"",',by Trosko and Chang. 1978a. 1981). The simulaneous adm,nistration nf t",oor more 'harmless' chemicals can. in some cases. enhance the b'ol~1

consequences by comulagenesis Or sensiliZalion.Exposure ofmdiv;duals ofthe same or different .pecies 10 the same potentially

h.armful chemical can lead to d'fferen' results because theK Can be a "ide ,-arietyof genetic dilTerences in the metaboli.m of chemicals to toxic and non-toxicforms. in the repair ofbooloi'cal damage and in the manlfeslal'on or exprCS$,onof that damage (Trosko and Chan,. 1979). The example of xerodermapigmentosum (xp) and albIno individuals can ,,,'''eto illustralethis point. Bolhlenl'hC ,mpalrmenls predispose tlko ind'VldlU-lto sunllght'lnduced cancer, In thecase of .p, sunligh'_induced~ damage In lhe DNA of exposed cells is notremoved. A,a result.lheseunrepaire:d DNA lesions seem toa,t .. substrates formuta,ions ,,'hich can 'initiate' ,he cell. or which cause cell death (ac'in, a, anindn'''''' ·promoter'. Trosko, 1981). Alblnos. on the othe, hand. "'ould ha,~mOresunlight-induced DNA damage unit dose 'ince they lack melanin ...-hich absorbsthe ultra"iolet component of .unlight in nonnal indiVIdual,. There appears 10 beno defect in DNA repair in albinos. How.....r. by saturatln,the nonnal repairs)'stem. some ofthc "'creased DNA damage could act a, substrates for mUlation.(initiation) and cell death (indirect promOlion).

MUlations ,.il1 al,.-ay-. OCCUr because of the in"itability of exposure toenvironmental mUlallC"" and of the small. bUl fil1l\e. ~hancc of .-rror-pronereplicatIon (re-palf) ofDNA "·...)·tiO\O a cell divides. An i""Kase of exposure toen,ironmenlal mUlagens WIll increa.. mUlalion risk in a criticall"ne of a givensomatic cell. If the orpni.m can compell$ale a def"""e Or deficient cell tithetby redundanC)'. melaboh~ coopera"on. temoval or .uppression. then a ,in,ledysfunctional cell "ill not ha"e a ph)'siolop:allmpacl. Ho"..... r. if by clonalexpan,ion o!that dysfunctional cell a 'critical mass' ohllCh cell' i"eached by theaforementioned mechanism of promotIon. lhen the organ and orpnism will bephysiololl~ally affected (Figure 4). The biolop consequence (disease slate) of.lICh clonal expan"on of mutaled or dysfunctional cells "'ill depend on the ..emcell and on the lene in the cell ,,'hich is mula led,

In effect, this hypothesi, predicts Ihal. as more mutalion, occur in dIfferentcells and genes. ~Ional expansion of lhese altered celts can OCCur in all our lissuesIhat are exposed to promoters. The Implicalion of this mndel i' that promotion(or clonal e~pansion) of dysfunctional cells" the 'rate-llmllinlt' slep for manychronic diseaSC\,

-­..-

:;..--...,a

_.. .•••.....,;;:-"',..(ev

._"'~ ._-

+~+~"m~:."0r ----".,.'0...--_"::::'o I- -- ._--" -,. ,~..-.~,__ ..._ .• 'h..~...~ - -~-

'~-'::':-C::< ==:'----, _

Fil:~re 4 l"ilo'lOoO 000 promo""o 00"""1'" '0 'hoe """SI' of <broo'" d,...... ooch ..can:onoJ<nesO', 'lhoe'oscle,...o.OO d,.bel••. A mUl.,",o. "'p<OOin. on lhoe J<oc.OO coil 'Yp<, ,rclo....Uy .mpl;fie<l cao Ie.d 1o. '<1;,"'.1' m." of dySfo"""on.lli,,u< in • gi~ orpo

g SUMMARY

CMmical$ can affect hlng systems b)I way of ""eral mechanism•. TM"ability to interacl Wilh cell membranes, "11osolic .llZ)m... and DNA art m.alUwhIch scnd a slsnalto adapt"'. cellular and organismIC respon When lhislnleranion leads 10 perman.nt damage of cell funclion. gen pression orgenomIC information (<<II <lea'h. gen. modulalion and mUlalion. r.specli,'.ly).,h. consequences Of"11010~iciW, g.ne modUUllion and mUlagen can Impacton the organIsm dependlOg on a van..y of facto.... Som. facto "'hich couldmodify lh. consequenceo of a chemical ..posure illClu<k

(I) th. cell or g.ne "'ilhin Ih. «II ",hi.ch I.J"lled. alt.red. or mUlated;(2) lh. Slage of d.velopm.nt ,,"'h.n 1M chemical int.rllC1ion ha' occurred;(3) genetIC "alUS;(4) nUlritional 51alU,; and(S) 'M ph)'ilolo8'Cll1 condllion' of 'he hon.

In .ff<:cl. lh. cau... of human t.ralog.n.sis, carcinog.n",is. reproducti".d}'sfunction. sene1ic and olh.r chronic dise.... are muhifaclorial. No one thing·c;,u...· 'Ii<$< d'l<:ase,. ldenllfylng lhe oalart of po'enllal blolo8'Cll1 harm achemical can induce (for mple. c)1<>l0~icity. mUl.1Ig.nicity. gene modulalion)"'ill be ohome help. Unde t.lInding the role of genelic. nUlrilional and ph)',;o!o&i-cal facto... in modifying lh••ffects ofch.micals in lhe hOSl ;, needed, Ho....ver, i'...,.,." to us 'hal .v.n ...ilh an apparenlly compl.t. und....landing of ",me ofthese variables. tiler. will be ahu}'. ",me other unkno""n and uncontrolled (andunconlrollable) facto... that ...ould pre,-ent a complete und....tanding of d,..a..causc and progression,

Quanlital;'-. "'limation of 'i<k from .xposure to chemicals seem, far aWa)'. inspit. of a belter cor.ceptual und.manding of ",m. disease Slates. such as theinllial;on promolion model of carcInogen......

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calcium on oorboo '.l"",blonde-induced ,ono;'y io OullU'" ral bepolocy1... Tox. ""piP/o(umm: MI, "41-212

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Domollof. L (1919). Go"no ..re,n","" promot"'" by' alkali", "'~u, p"rild-"'lb'p«iol ref"",no" 1o bil< .nd other .u,f"'.n" a' promote" of po<topera ..,.. pstnocanco, !o/rd H)'JI'O'~""" 5, "63---416

E""nt. l.. and C~rut". P. A. (1'1ti I). Tumo' p"'m'''''' pbo'boI-12-rnj'ri"'lo-lJ-Oc<I.toind""'" obmm_1 domaJ< >i. IOOIT«1 action, ,\'a',.,... 293, 144-146.

EIK',"",o, LSa..kl, Y K.nno, y .••nd y."....ki. H.{1'1tiI). TUIOOrpromoto"cau...raPId .lId ""'<n,bl' ,nh,bi"on of lbe form.t"", .nd moinl<naoo: of .1<011'1001 «11«>uphn. in oultu«. p,«. "",•. A,4<I. Sol, L',s"l 71, ~8-S63l,

Fobmy', M. 1...nd F.hmy. O. G. (1980). 10t.,,-.n,n. DNA ;ns<'tion, ••d ,he al,o,"",," ofI"no .'pr-.s"on b)- cam""l'0" C""", fin .. 40. 3374 ·JJS2

F..ber. 1. L (1981). Tho 'ok of calcium in «11 <loatb. L/, S<i.. 29, 1289 -1291Fialkow, P (19'4) TIl<: oripo.nd &,·,101""<01 of human '"mOn "udicd ,,"b 0-.11

mark,,,. S ... £ltgl.). .ifNi.. 291, 2" JlFould,. t. (197~). -"",!,kuli< lNo<lopm<.'. Vol. 2. Aoad<mio Pr.... N..., Yo'k. london'

129_,.Frei. J V (1916). Sonu: Il>«'lGon;sm, oper."'" io catano........, A ""';0" C~,"../riol.

I.It,..., .. U. 1-2~.

Get,... t. E.. ond N••I, R. A. (1981). MutoJ<ru<;iI}' t.."n,of2.J.1.8-l<traehlorod,hon<o­p-dioltin in butldi." OU.OlrophJ of SaI"""",I'" ')'p~imu'i""'. Tox. ""pl. PitnrmiK.. ~9,

US 129G'ntil. A.• R<nault. G .. aDd MOTlol, A. (1980), Tbo 0!T0<1 of lho tumor p,omo",r 12.()_

l<lfad<anoyl.pborbol.IJ-.....l< (TPA) On UV- and MNNG-ind..:ed ..."" 01110­

malld "..lGo""" in mom"",lion «lis. Int, J. C"""'" 26, 117-'21.G""tttle', 1(., and l.o<h'k', H (1916). D..plaC<ntal ..mo."......", ,notauon .. ,th lbe

<are,no,.", dl""'lby-l\,'nuolh ...."" .nd u<ethan< dunnl f.tol hf, .nd postnat.1promollon .. Ilb ,be pho'boI <>ter TPA ,n • rnodLIied 2_..... 8o",,,blum Motlr.",oxponrnonL V",*".·s .1,<h pa'•. A""" His'oi" ln, 29-33,

Gol<l""n. O. 1J".nod ClHn, J. H. (1981\. lnler.won of <lllan'" ...,,~ tnolol"'"'l mem·b........ F<dIt /'r(><, ...... 207)-2016.

H.hm•• M, (1971), Ep-odemiolo"" .,'Id<nco for mull""F 'heory ofcarcinos<"" '1>'. JCanN'" 1. lS1-56&.

Hoanl, V" Chanl. C. C. aDd T,oUo. J. E. (1983). Aphiditohn-IDdllC«l <Ddorcduph·cotlO1l in Chi.... ham".r «Iii. C"""., 1IL,.. .o. 061-136&

H.d.r. E.. Fu..nll. N. L. KUl>L W., Mark>. F.••nodlllitlmann. H W, (Ed•.)(1982). C...<tU<iIoog<""u aNi BioJogkal tj[w of T"""" l'r<Jfroo".s, v",. 1, R...... P.-..., NowVo,k: 66& paS'"

H<nZl><rJ, E. L. (1980). Biocbrnucal.nod immunoloJi<>l.I'I'''*'.... '" I!I<: .,lldy of lOPjunc<i<;mal comm.nica'ion. 'I> Vi"". 16. IOll-I067,

H<>pl::"", R. L.. &Dd Goodman, M F, (1980). O«>.)'nbon""l<ob<l. pool>. _ pamnl.and soqU<1lO< <onfilU,"t>on aff«;1I.1 bt-omodoo.yuridl.....nd 2·aminopuril><-inodu«<lmu~ I'r<><. "'lin. "'e"". Sci. U.S..... , n, 1101-110$

Km..lIa, A. R, (1981).ln.u,ipHon of'l>< .If«ts of'l>< pbo,bol"le,TPA onca=""Fn·indu«<l f."....rd muto 10 ~'h>OlU>nl ....=">nc< in '119 ChI.... hams,.. colli.Careiltogt",sis, 2, 43 1.

K,....I... A .. aDd Radman. M (1918), T.""" promoler inod ..... oj".. chromatid..chan...: ..1.....1>0< 10 mochani.m. of caranoacnc>i'. /'roc ""'I>. AeM. Sci. U.S-A.,75. 6149~I~J

Kirk"OO. O. ,,, Smith. K. L" aDd Abb<. N J, (1981). Failor< of cblo'oform '0 ,,"'''''''cb,omosom< dalll&F or sisl.r cbromo'''' ...hat\J<S ,n cul'ured human lymphoql..aOO failu" '0 IDd""" ,,'~on In Esl><n<hl& <oil. Fd C~,. T,,~ .. 17. 6SI-(,$(i.

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l.ocoo·<IISInn. W R (1919). JUo<Ii"""l In,.,..Uul.r commun>c&,ion.nd 111< <Onlrol of&:,o....h. B;«.iIrt. ~y,. Ac'o, 56(1. 1-6S.

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MooIp,br. S. H.. Day, N. E...rId Stevens. R. G. (l980} Two-.toF mod<I fOf com·"""'....." <pid<mioIocy of broau conc<r In femaln. 1. ""'I>. C_ I." .. .,. SS9_S69

MOI1rUIl, J. C (1<M4). A des-.Iopi... f..,0, I• .,;peri",...lal bla,,_i. J. Po'~ /Joel~ 1&1_181

Murr.y. A. W. o .nd Fi=.hl. D. J. (1919). Tumor promo'm inhibit m<1&boIiccooper.'ion in <"""lIu... of ,plOormal.Dd 3Tl <:<Ila. Bioc"'m. !>iopJly,. R... C""""""..91. J9S-lOi

Napo. M.. Vaha';. T...rId SUl'mUra, T. (1m). Dilf•......" in .If«t.of"o,hanna" "'"~

....ncu. do.... of ,hem...1 ,"u'"l<"' and amouo" of S·9. Bioc""". biop/t,'" h,.C"""""" .. SJ. ]73-311.

N.p....... H.••nodlill"'. J 8. (1979), Eff= Ofl"mor promol.... prol.... inhibi'on&Dd..pai, p"",",,", on X.ray iOOu«<l ".w ,~ro ....lId ..cllanF' in "'"".. con•. I'r<><,naIl>, "'ead, Sci, U,S...." 76. 1943_1947

N....bold. R. F...nd Amos. J. (1981) Inbibition ofJD<I&bolit<oOpe,"HOII 1><1.....0«11. in""It... by 'umor promo..... C{UC~su.2, 2.]-249

N....,.lI. G. R.,.rId Ellison. N M. (1981). /'r~,.,. ill C4IIUf R".o.dl oitd TIIt,apy, V~17. /l'wtd'i<I<t oitd CatHYr: ElioI6fy oitd TmJIm<rI'. R..... P.-..., Now Vork: 44$ paaes,

Nicholson. 8. J" H"nkapill<'. M W. Gnm, L 8 .. Hood. L £.. and Revel, 1.·P (19111),R., ~,... lOP junc'uon prol.in: properties aOO portIa! "'ltl<I>C<. h9c. tIlIl", Arod". S.tU.S,.1 .. 78, 1l~-7S9%

198 M~,1uxh fw £.<tunDling Ri.<k ofCkmical InjUry

N.,olson. G L (J979). Canctr mewu~~ Sci"" A",. UO. 66-76.N"'obon. G L.. ar>d Cu".ad. 5, E. (1m). Tumo. mel.l....... " IIOl d"" to adaptation of

~1I. '0 a ........ o'pn .'""""",.."t. Stit>t<t, 21!. 176-178.i'iO<lm",. T. (1977). S,m,!anty of ,he m«"an"m of <hemlt&l a.ranoaen·,nit"'ed

"""olmesi. ud ca.cmoFoe>i.· C""",. IUs.. 31. %9-973.NoYo'eli. P C. (1976), The tloDal ""oIut;o" of'OIDOTtcli popula'i""•. SNmt-t, It-.l. 23-28.PaNY. J. M.• Parry. E, M.. and ~l1'tl" J C. (19&1). Thm<rr promo,«. mi'ot'" .""uploldy

,n j,• .,t. No'"",:tt-.l. 26)_26SPltot. H C. Gold,..'ortlll. T,. Campbell. H. A.• and Poland. A. (19801. Ouan'il.lti" •

...-aluahon of tilt promo'ion by 2.3. 7.8·,0'1 t-..:hlorodibntzo-p--dioxill of btpo ,<>cora",,",.....,. from d'e'hjinnr"..m'"". C........ IUs.. *. 3616-3620,

Pltot. H. c.. and Sinca. A. E. (I'ISO). The ....... of in"...t"", and promotIon tnbtpolocora""*"....... Btot/!im, ti<>pI<}'s. Acto. 60S. 191-215

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Poland. A.• and GIo,..,. E. (197<J). An ..lLmat< of'he maxLmUm ,n ....vowvalent blndinlof 2.3. 7.8.,<t.-.chlo.o<!ibenz...p-d",,,n '0 ",t ti,·., prot.,n. ribosomal RNA.r>d DNACQII«' IUs" 3\1. J}olI-J.l44

P",,.. G..•nd f"ldle'. 1 I. (1980), The pa,....,......."of a.no:, ""'.~r.a.... No,"". 183.139 146.

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