I~ I Y ·* i I· 1:I i , t·l~ d 1' li· ·1. ,, I :I, 13: I ·i:·r ~· '' i,: I :' '` I';r .~t~:~l~d;RII~UQIIL?PY~ II I · r'; *; ii * *. J :v,j ~·I r I~ ;I Es O FO ~t~3~i~B~E~ 'ga~ ~P In \D ;.~~mmrUIY···Y·---i~-~pu~~ __ ,_a '· b 03 Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Awiy r~'pd , ~· ' ··· ~,·~· ~ ~~l~i(Y1 '~~S'p ·~~a. ·1 x ~rt:t~?p,n,!~~,~·' · ~ · ·- *· "~~?~~' ~· d . m: ~nY~C "' · ~rs~~~U· ri· ~·. :~" ; ·.· ~: ~J~·. ;e ~··· yll W.p~. ~~L " ' L· Sb: ' '~ ni ~r~ · ,,ka"~· IRBIR Tl~b UL~) FH RESEGRCC I~BORliS~u HON~RE~ Thr OctrL:'~l of ~~anenie ~~:tv~:l sl Lhr~i· ·~' IL·· ,,,,,.. _,, ,e g [e S InC 031[CO S~kC In dterld SY· :~· LYnb ~bn~~ er: 5, sc -·'/'~' ·,a: re3b 1 i, ~,~... . !27~E8I~ .,J i. ~;.·~e ~n i $d-· · .~~~4·~bZr .,,, s: ·: · I i...,.,, ; i; ~b·.i' g0 · i · · '- ~· ·~ · i r· I: I~ c I~ ~~t- ·'· I ~? ~I * 5 - L ~I i i~·~· ·13~ ·;· ·- I·? 56~~~~3 :3:i;~ REW*~rc~- ~~jlmrrr~rr?~FI' ''` ~ k: n N ;u~J:·: ' ·~·~·· n ClibPDF - v~~fastio.soni Aesecrc~ Laboratory aeoor: w:. 150 631,1' No, 1? :nDI!IAL T5Bbtw L!K1T~C RESEARCH :3La u lk60n4ibRr M~N'TR~! ~b~cnoH OF nOiAu"lHle Acyrr:rv oi H["IC:S MIO TCSRCCD SMOB in A 8~:iEliA: SrSi" II : F~lli%r,a: Proier~ No : ;-7i08 !SSVEP BY: Or.;,l, Smith O:~ ISSULB: ~e:em~er 2!, 1911, DISTR6GiilOH I, i~~raty lj~ Dr, G,G, Fel;on 2, Br,R,R,Glbb tS, 3:,F, Harl~e 15. Dr. I.W. Hug~er a' D~, T.b, Snlth 16, Or, E,A, Sasfcrd I·.,~ · ' 5. r,r,S,El,tandllsh ii, Or, P,,k. Sanford ::·;:I;*· i, Dr,S,J,Green lg, Mt.R,G,HichollJ i J:l 6, Or, 5,3, Green 20, Dr, 6.3, Ounn 9, Dr, S,J, Green 21. B. C,,F, El Naggar 10, Dr S,J, Green 22. a:. E,R, Fleiesld!e ii. DI. i.J. Cun i'. Y3. ~I. ~Dhri~l ~· 12. Dr, D.G. Fclton 24, b, i,L, lit! 25, r,r, E:,n, Schetsak ·:~:: .. ` ~:~~ .. · · TV ·~ ·· --~·;· ~ · h ~ · '';' U1 .'~' ' '.· \D Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 C b ha~ oi axi:~H7 ~ L~a 3 Tobacco S~ke in a Bacterial Svltre SOa~! The bacterial test described b! P,osenkranl's group j:a~;!· ~lr., ~ (7), 970·i3, 1971) and involving use of an Ircheri;~ir csll strain deffcient In ON~ P31~r~lCI was used for the oe:e:::'on of ~~:agenit activfty of tobacco smoke as well II Of some P~r~ :;n· 13un:~ known to occur therein. The ··lflnal ~:h3~ :descrl~e: b,v t~e authors was found suitable for Ce:ectinf nuh~enic ~C~rl~. of p~re c~wic~lr, hut failed to detect mutagenic ac:ivlty of t:II:c1 smoke. However a modified y:ecedure, Involving pre·!n:~:r· :lon :f tobacco smoke Io1Utf oils with the bacterial cul turer I le!:ie~ detection of Rutagonic activity in cipare::e, cioar and pipe I~:il!. The oil of the prl·lneubatlon mixture arkeCly influenced the e~· ~9?slcf~ o:;he t3blEec smoke condensate, which was more 1C~v~ It alkallne than at neutral pH. The bases nicotine, pyrldfne ~~d Ordl~lll rln ~~UI Iltlprde coly:r~ P tha lo~e cmlacq anC shoMed activity at pH 9.0 but no: at neutral pH in thl ~~ ~'*~.· cen::atlons tested. Cyanide, ~droxyllllnl fo~~ldl~dc, acro'.efr,, hydrocuinone and pyrogallol were foun~ to be mutagenic in:~l system. while caffeine did no: inhibit pr~th of either the nu~~: or the parent culture. M!:aS~I~ activation using liver ni:rorc:~i ea:pel was no: attempter, I: Is fcl::ha: this procedure oe:f;! fur:ner investioetion for routine ~~tCrinp of tobacco pr3:U:: JM~~~ea, :t;i 0: ii a ii o a ~· Ci, ~ ' · ? ~v ClibPDF - v~~fastio.soni 6' r~TRoovc;Ion Rera: years have seen a renewed interest in nu:aSenici:v :ertr in Ei:a:Smirn a ~eAallr prpCir:sn a: eariirP?!airitv in mananalr (1-31, The availab~li:y of sue~ quick ore·l~r!enine prJceduret would :?ke 1 cOnsidereblr load off aninll tcs;in5, &nd Prove of S:e!: value to the ehenical, food end drup indl;I:rie:. ;ne teaaeco inb:~,:lo, would gain e valuable tool, both for r:ueylnj Cndu:: eC'ficr:ion Lr well ar far tracinf nuSgtnr anC clrrible czrcinopc·s fn:Bt:aalex n~ixture !br: is tobacco sr;olie, IwO :e::e:iy described ba2 e'ial systems, describei ie:ae li:era:1:e far rC:i! Cete::f on Of nFtrPeni~ and p3sstbie c~rcinottni~ ~E~lvit,v, are ~eie; used in thif leborctory, These are (1) ;he p:hod drr:::!c( ?v Ilaer and hlr 6?ius (1·!/ tsaloyinp hil:ilin! 1U"P:m)hr o! SLI~EILlil tvDhllUfiy, In utieh reaniani ~e ~r. tl:rophy are lunl, and (11) :he n~thod dolcribel by Acres~nn~'r 9mup (I,B) dgvina a Dib Polymeraie dlficiest nutan: of jrrheri:!ia L ~·i.t ··~i:' rpM (Pcl b'j I itr plnn: culture (Pol I'). In the latter, 1 ~~E:LT inlili:iSi pf:he a~utanr Log~rc4:: the plrent cu!ba ii ..,.r., used a a criterion for nu;agenfei:y. ihis pmcenCure has beer: I: used ex:elrivzl? id this laboratory to fine out if tObCCtO Smke, as *: well as 3Cns E533Lmd! known to Occur In I:, porscss mutagenic 5 a~tlvl:y, ii~iJ J:e41 and the results obtained, f0ne the lu;je:: matter of this re::r:~ : :,"'t': · ;- · ~i~i~i: ii ;r~·· lu ·it a i ul st : ·"·1' Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 5; ri:C: ~iR~~1S II~ ~E~ODf I fh~ adla Mpia~C for the aafntenanec of D~e:lrle~ I:r:inl t ~~~· L. arwall u ~rper~~ll·~e taS by lelJe aadln~r:=lj· Frondurn, hcve ~e~ Idquat~y Cesorlbad br Ro~~n~ an: ar e ~:~~· 6 ~" ~·norken (7,9). ihr pn:cdure ea~~~~ ~~eJ w~ a~n~~l! d3LI t3 dn La~~l b Rna a d, (T), whill ~( ~=~,, ·1~· · In~~lnp pn~n~~Ucn of edtunl wRh the trrt ~~~nec w~ Jinilar to bit Ile( by Lnar a a!, ~d lp ItPb~p Le ~~dt h of lfl?toxin 1 O Lt·ldh gH·~tl, IJ noll a to t~c: 6~ 4~if· :~&d $ Io*ar.t hli for rtuCyinr Ic cffce: of ~c~o~a i ~lpoa~) El~l rmltart lelk~l: eD11 P~~nY. I~ For pfcp~rstiC~ of llnokl e3ndc~lattl, ooronor:iai!v ~rli:L~e t · c!lcrcttel, figarltlel, and elgars mre lmokcd undor I;~ndr:: esn· 1 ·.~ C~II o~~nlb Ielao:rd to ~l~lh Lal dpn=! I~~F f.: (1~1 s~l 95 d pPf~, of 2-locond Ilrl~o~ Pba overv Ig~ ··· · c~n~l~l~~e a~~F lehfne. Io ~e aJe ol ~~ I~~np, a ~ ~1 of bl~eo~ duration uJ oollaotad ovary 10 ~~~1, ~d ~e ts~l~al wora !·hd in (gn glaPr~a b IBl~al ltaal 113~4 Flpsl Ind I~t~inF 1 pfpe tobae:o lmoklng maohior deripncl Is :~II lrac:atDry, me IRo~ war trappe~ In IPfrai con~mr~ cod~ ~ ·~G"C md tho Eondc~~~ wal dl~~vc~ out 8lng ~~~~ of 1~~~. 1 ~i s" ;. ~_k· n' ~'~~ "LI c~Wnrrd off InC :h condr~a~ drioG tc ~a~~ w!lFh; on 1 ro;lry cv~PoratDr, waifhed InC rc·dl~~olvc~ fn Ivi~~E I~8P p: oootora, I ,~ ~· ~39 · .. O ~C' 3~'·. N I~j~;l~C~ -I~··~·::a :I C· R~! ~; ~ra:E ··c~:~~; ~I ClibPDF - v~~fastio.soni RESl'liS ID OISilSSiCI ' ··.;';I::I· · That the plate method employing filter paper discs is suitable for F~re compounds is shown in Table I, Chemicals such as ar~icillin and chioromycetin, reported to be negative by R:~enkrcnt (1,131, show equal inhibition of both strains, while rrrrtal violet, I!~?1Re:hanerul phocrl·, rqpr:ed to he pr~tive, show a greater inhibition of the poi X strain than Be parent ·':S~: culture. from be table, it will be sea:hat aerolein, which ":"'". is known, to occur In tobacco smoke, gave positive results, While -~;5~ . k the mu:rEeni; effect of acroleia is toown (l,lS), there results E· ·~· . s~;;e~::hat tlls compound be rc·cvrlurte! in carcinogenic tests, :::: ~·'·~ bearing In mind its high reac:ivitl~ Caffeine, as will be seen h f.3m the table, diC not inhibit either J:r?in, although i: ha i ;:~~::ii;:.·· been re~3:teC to give positive results ic:hls system (2). Co~· .·.~~·: pounds such as h~l:oSuinone, pyro~lliol, which are au:coxidisl~ie, mi potassium cyanide and hydroxylarine, which are unstable in Frtren:r of lit, C02 3: m3jltC*gl le:e also exreined anG there gave variable results. These have been examined bythe pre-ineu- Lttion proceCure,the results of which Hill foll~l, ""' "~ Our main interest in:his test lay in applying it to tch:~e sr3ke condensates. It is here that the lisc procedure failed to work In Our hands, ever. though the method has been applied to eig· rte:te smoke condensates by Rasenkra:!: (13, personal ea~unie~:ior,), :te procedure initiall? pale I;ignly vrtidie rd In:onriJtcr,: results, and we thought this to be Cue te ehanocs in the pcl F: r:zln. [oF.le?ven:ly, we e~sined fresh cultures through the kin~ e courtesy Of Professor Rosenkranz, as well as from another labar~s:v. These cultures were maintained as frtl~e~ permanents in Slyeerol, tan~lna:ion of dgrre:te smoke non ene rais:en: results an~ showed ciearette smoke to be negative as will be seen from Table ::, .~i~i~i ' ' a ~~~r C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 G :~5!r,:~e examination of e0ndeesatCI frs cigarettes, cigars Eel cigarillos as well as from Fi:e sn3i!ing also gave negative results as shown in Table III, lir;ct;lsi:fve results have been np3r:eC for cigarette smoke by Rcseshnt and ~nderron (131, End since thr nature of tobacco smoke r;loyed by these workers vs unknown to us, we prepared smoke fractions by the procedures of Elmenhorst and Griner (15) and Swain ~~ (16) and examined them for mutagenic activity by the Cist procedure. Once again negative results were ob:a;ned. The nefa:ive results obtained with whole smoke as Mil as with role :rr::onr are no! rurpriring in vlen of the water·inroluble 1~·;:· nature cf t"ese c3te:ials. Along Hi:h eck ~ef diffusion of most of the smoke p:nrlEl, it would aPPear that 3emlbrllty into the .,~~,; bacterial cells was also a problem. We nan employ a procedure 'fnvotvfng pre·iccubatlon of suspensions of the bacterial strains (·:dth rc!:o:!·eos:aining rolu:ions of :ot,t coade?rater or with ~. frrctieas el;alnrd berefron, Employlno such a pre-incubation ifi~I:i~~ep, we hcve ou been able to show the: tebr::s aoke condesa!er i.·;i·~.'~·l~i~·:,···:·':1·~ I: obtained from e'FEre:tes~ cigars and pi~c·sms~ing ate distinctly mutagenic in :ibe E.coti system, This is clearly shown in Table IY, il~r:: cl garetr smoke appears to be less mutagenic than cigar smoke :but more so than pipe smoke, however, more experimentation will ii·~f;·"~;: ··.:··.. be ne:rrra:y bei:rr such a Ci:feren:11:ion can be shown conclurlvely, One of 9e eb:relts in these exoerimer;ts was reclval of water from the pl~e condensate, not to mention the difficulty of working with highb fnrpiuaie ma:etill in in iFveous envi:,rt~n:. frl:~iOn~tilE Of cloarette smoke, as carried out for ob· : talntsS nitrome:nane fractions of smoke (15;, has shown activity ·~ in bo:~:51 r~:~enoi and ni:ranethane fractions (ia~le Fl. Since a Icrqer;r;)l::ion efthe smoke fraction Il:oe:a:tated In the methanal fraction (IO·i5: of total dry condensate as against 9·10: ~c~p~· :, ·~~~ ;:I~ · I'.~· n· ···· ~ i. . vl iU~. ~n ~ I';T:'' ·· Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 I OIi~p activity, vhcre!l In :er;r of rpe;i:it ac:id:y the txa fn:;ionr may not be different. Insufficient data do not ptnnf: su::! cal· FUII~IPI involvlng Ipeci:ic activity, I: is also parl(ale that i· · the n~tro~thrae fraction, Nhlca should contain most of the polycvcl': hr~recar~pnr of ciSare:!e ~s~e, needs lnetabolic aetlva:ion tPfore 1:5 full mutrcen ic potential can be me~~ured, i Hrvine shown that a Ire-lncutatfan step ir necessar:/ before t1. e~t~ining a Plsi:ive n~:lon for tobacco snake, v ;boueS: if I' worthwhile it r3 see how 3ir Procedure xorked for pure e~aicals, Particularly :alre, like ?vCmxylaminr and cyanide, the: Ce:Jqose F· rapidly In presence of cir, t02 0' noilbn, iroa Table ~I i: nill be seen that a;~3lein, hydroqulnone and pytogallol were Positive, in as much as all three f~:aited strain Poi A' more than gey did Poi A', Caffeine anE ai:::'nl, even though exlnine0 a: fairly ~':'*'' high con:en:n:ionb, f~iiel :3 inhibit either strains, The detection of nutagenic rc:iv;:v cf jyr3galioi is ia:erer:ing in ;~ liFn~ O! ... ; ~ I report by kdeacn anC :ir,ney (17), stating :ha! pyrogilicl Has f· :he art ar:ive al3nf phreliie eoqlvndr tested In pr0eucinr :unourr Curing early Nlco:fana hy~ri? sadlin~ developrcE:, while e~er;ot and catechol were In;::ivt. I: is eilo inten~:ing Llourr ru:o- oxidation of pyrooallol is known to result in the forna:ion of ,~: Ivleraxlde anloar (Is), r5jth in turn could result In the ~ern~tion ·::~v~r;c~.· i..: ::~~d: .- Of hyd~og"n psldde end tie mit Ps:en: oxidan: ~nor, to Ill:ir:ry, tie hydraxyl radical (19)~ The results c~:rineC with pc:assium cyanide and hyd::xyladne r:, arC presented seoarately. iron table Yii it Nill be seentna: 0o- ~j~F~~i·~.:.: trsrlun eyanf de Inhf bits thd mutant, Poi A., more than i: does the 'pannt, Poi k', While Table I'I~I shows that hyCrOryllrE~ne has the lame effect, Usile the a'3cenic activity of hy~roxylarine i! wetl·kglkn, that ef cyanide war a surprise. ~~t?e_mu'dpenl~fty of cyanide remained unde:eettb on ac:nun: of the ~lto~jdty of this cqovnd. R,nevrr, S:ltr and [as:lr (211, 3 C~ o 1;1 ·~:. .\o cn Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 rl I~rly~l 1901, FrpduaC nlplrc:prv ?I'I:f~a:lu:lntr O! ynr: by Ix3esun to cvanide, hr:her stuCier hive I$own h::~r F" of the p~e·incube:iol ~x:"e has I arkrd effect on RutaSenic activity of tobacco amoke, ~ Ys is shown in Table IX, A: pH 8,9 ciEE:e::e snake condensate, :'j. ii at a concentration of 1 g, pt: mi, rec::iln dnture, shoxr a '"l""""~rtill ini~l~ I: ibinlii:nbi. 1I1II: 1 ;pH 6.8 :he inhi5ltion II nlglidtlr, Su:l i D~ effeF: would ruSl!r; 1 that brrle coqouodr in tabaea rlKI$ are al;a Inrolvtd in the ~u~5eniefty of tobacco smoke, thus, when nicotine was re·exasin!d f. at rtkaline pH values, it showed a Cif:er~e:frl inhlM:iPn of YII ::·i~~:':~ ~EseMltrainr, an Inhi:f:fon no: Evldetl: ~: 317,0 at the con- ;I' Cen::E:rons used, this will be clear frz i~?ie X, irom figure 1, which is taken from a paler by 3orls, (?1), it nill be seen tha: ··:·r~ there is very little un·ionised nicotine at Fe :,O, a: Which ph most of the nicotine is eonoxotona:ed, At FE 9 most of the ni· lo:ine rili be unlonlrll,lhl:h c)jaen:ly 1~ ,,h, f~n in xhirh It is in~f bltory to E~col I, r: must be p3iefeC out here that in ' CD":'1S' to cicare:te snoke if which n~u:1Felic activity II dete::el a: 1 Pg (eP lets) pt: nl reaction tix:un, 5 q nicotine per mi 1:;rlM:... .Re:laa sfxture and lancer p.re·lneuSa:iol :fa were needed before mutagenic activity was detected, Se! 5a?le X!, This is a Ilrce unount of nicotine when it is eonrideteG:h~: 1 mg of condensate jl from regular flue·cured cigarettes does not contain more than 60 ug of nicotine, which is be far the major eo~monen: of the i· :;::;::I·:~f i tarle fraction of tobacco *al;e. Ihn, thr Mtagenle clfec~ of nicotine is, a: the rr.os:, very sliEh: eesareC to that of whole j. condensate, .. ~i~2~r . .· Pyridrne and pyrolliCine show a similar d~fferentlL1 In· ~r t~. ~, hfhftfon of the E.eoll Strains, and this inhibition Is observatie PH 9 but not a: pH;rl wll~ be seen in;able XII, Again, like ~lj~i:i:qt~~r.lcotln(, 1,,,,,,,,:, are nrtded to de:l:t a:ePmle ar:ivi:v ~fr · f·. o N \o\o·U1 ~·::· Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 4" ani when l~us:I of there barer that dc:lr in dar:et:l mwke rr! conlie!·d (& aawun: of lyriClne dl:e::td In elpnttl r::ke is a~31: one hundredth that of nic::fna), it is clear thyi. :Lele bases :rr, a: P10J:, only weakly nu~geaie ecqaed to cic· :~·5~~~S~t~,I··~~ rre::e sG;ske. Of EDvrSel th ~e results sEtS!s: thl: Other nit:3len· ·: Ns·t~:;: _·'ib'·: . an;alninF ~:~?oun?r known to occur fn tobacco rmete should be Ixdned f;::hPir mutaotnle activity le:he T.toll system, r~· "~1;~~~·^l~~~iil~Moi.lEiYn*Uby i.' j ·. hlue fcund gl: S·~llnurl~nn TA·1538 Ir tntnlely IEnrltive ts '~:~~ci !rJ9an·c:lrni "O Polyyelle ca:lund~ EnC relatively inlenri- iii tlw c ·1L··l ~I* ,,,,,, potycycllcs, j· icr:'er I:dei are ~n pro5:erl :: nsiie the P~3cedure i' j~\.:·: rope sens::lve end qvln:i:atfve so that it si;:uld C losr~ble 5 Clffe:m:1l:! sske from different smok:E; pr:!ri~lr, Ile:E~olic t C:1·~; ··: i activation ullng 9,000 g suPerna:rnts fr~; lung an: liver homo~en~t!s f:3n dlfiere:: aniPai rperiel will llro te a::em):tC, lin~e tobltn V~r~t~:··; · 150kC COn:liEI Ecrefnogas that need to be ae:ivrtel before they '.C detee:e~ as ndagens (5,6,22,23). b::en~:r are rlro being tade to dilaer in to~ce;a smoke, other eoaesunds loslersing v".: ·' '' ' "'P~genle E;:lvl:y, in e:n:lusicn, 1t may be stated :~rt, presently, by em- I ploylng a Crr·in:~batlon step in the Rose~trae: test, it Is possi~ie f : · · s~ daonstn:! mutagenic activity Irl no: only lure eoe3ounds, hu: also In tPSr::o smoke from dffferen: tinb of rnaklnq Products, as well er in IMke fractions, and :bat:Jis;!:rot~ill pro;edvre .1 hrr ~o:enYr; for he In routine wlitorin~ of :s?Ecea IrPduet i rtc3tfiertior;, SC~'cl~B.' ·-····s· .~··~·· :: ~f~l?·.· .:: · I:I?·f~Y1~~): ·I O '"' · ;s~~*t~ ··· ~ii~i~i~ .··~·~··.r:;~ ·; Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 "r: 5 9! REiT~INeES i, I M~L!n3ER jeC.) C~zlc~ ~u~gcn~: P~nd~n Ind M~hu for their Oet~Etlon, Yell, 1·3, Plenln Press, New vp:i, L~I 1971, 1973, i~?_ 1 i, IUiaS Ed,) e~lagennlr tcstfog ale~akdl, b: Press, Inc,, tleveland, Ohio, 1974, 3, The TIs:lng of Che~featl lot Cbrefnogmlcl~, ~~t~PenlEi~, ~:Etoge~lcf:y, Heal:h and W:lflre Canada, 1~13, 4, B,N, bES, In "~~Scnle Effects of Envitonmental Con:mi~r~tr", :1. 5UTT0N and M, ~ARIS (eds,), Acldcmle Press, N,Y, 1972, ,a;P: L U, a, N. B 3 LL II·. k~ Y. hL L, ~IS,) ~1 ;821 1973, [~~:: · 6, B,N, ~~iS, V,i, 03RSiDI, E~ Y~SAKI and F,D~ IfL ~, ~, II ;2;1, 1973, 1'·~ · E.E. S:~;iff, n.o, ~DERSON and H.S, RCSiN~~Z Cancer Rls., ?i , 970, 1971 , IQ~~R LJ. ~SII·lt, I~.:;PI It e. ~IW, rP:~a nFsf~ Res, Co~n,, 4, 526, 1~1. ,~~ 9,.H,S, RCSENKR9NZ, H,S, ~RR and H,M, RCSE ~:k L~L,b·C·L R.e, C;RRER; E,e, M~LLER and J,I, H11LER. Cancer L1,1~ ZP5el 19'72, ~~~~·"'11, H,S, R3SEN~N1~ tbld, 33, 459, 1973, 12, M,H, EILINORIA and M,A, HI552T, Bcltr, tur T~~a~~r~eh,, P, 27, 1971, 13, H,S, I1SfN~~i and N,D, ~NOERSON, J. Istael B1Pchem, anC lie~blol,, i, tO, 1973, 1$, e, ~c~n and R, ~~ERET, tomp, rend,, !65, 1799, 1967, 15, H, nlENHORSi In~ G, etImER, 2, Krbb~forlch., ~1 65, 14fi, :· ~~ 16, A,P, S~~TN, J~E~ C001ER and R,1. S~O~H, Cancer Res,, ~, 579, 1969, 17, R,A, bl31ASO~ and 1~ L!NMEI, Cancer Res,, 31, 2150, 1973, ~~'7P, r C'~ "' ~~"x: i L O ~,~cdlP.B 5 C N c114 d~b~' ~; ·L; BsS~ ClibPDF - v~~fastio.soni I·1 I_·· '" 'e i· :t~ ·' ··;i.;l ·,r IX;:"r_"~' si i ·'· · i. :~· Ij ..~~ .,.,,., 5EbFI·' 15, ~ 5, ~RPUIB ~nl G, IWRXLUIIB, br, J,BlbcBa, ~· ... '· 19.' I, RIOWI[H. 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" ~.:,·.· Oi KC# iiOW TIPIE;·:- RiACT1ON MIXiUEE ~I MIXillRE ~ 1 I I POL R' 'J A' ~!~ R~trroa (Aa) ::: k-,:'~· · ' i· . ··:: ::' · ·: '"' 2,5 i O Nil 'U11 5~i(i~ 101 0,5 1 NI1 250 ·.~rnu I IIIL llI1 " ' I;~tts :"~:· I':·:~k7·· '···' · · ·~ ,. !,O :i O 148 FULL EOWTH 101 · FULL ~7,aiilH loi ···IL ·· · 0,5 NIL SL:.?i;Ct.II:: ···t~':''* I~· RLI BWIH 101 "~" NIL *1:F:3 ~· .. ·.. I"·~·· 5e '.· 'l···u ·'· ·' "' n· rtr ·j ·.·-' ' "·· ·;.: ·: 5$ X 105 ·· :· " *;""·1 ·. O · · '~` (Al 195 X 104 ,vl~·:·-u. ··,, . '·· · ~· 015 U~ X 104 200 X 105 '':' '' ·· I .l,f·:: 52 31 R 10': ~ .. · *·~: ·.c? ·· " ~)· :":·:"~ ~~j~: ·' ~·' 'a'.:' ··· ~~····'' ··:'*.·~.·· :O (8) 400·' n]L1 E~3WTH 101 ·I ~~~: :~ :.. 0,5:it: ·· JZj FULL E13k'lH 101 i,.; ~-li~i~z · ···· 1,5~`·:· 52 :: FULL 53WTH 101 :'I: . · -;···· ..··· ; " " '' '' ' ·`'" .·~· " o ···· ns`x lo~ 10:25w:': " ~ " ' ;0,5 106 X 107 U3X1jt 1,5 :..·i.·· .. ...; c 12 X 107 115 X 167 r;· 13 · ., ~ .·· 1 ~ ~i~·:~~ · 2··1· ·:··· Is~ P i~·:'·'··. 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