Document MMaw856vgj8OjRppZw7Y5RQ8L

R&S 108269 sJo-medical kxs larch DOCUMENT DESCRIPTION PORK 63___________ 68 69 76 Duplicate in all cards:--> year as-1961- File number I Fight justify [Numeric only] Author (s), as Last Name PS (No Punctuation) and coden for journal as JAMA preceeded -by,one blank space 7/_____ 73 L/C-- Sub-Index Cods Title of Reoort; end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with -comma-space. Avoid other punctuation; do "not abbreviate. 12 * 61 '62 !:___________ ____ Cx--:Y- i4* Uc_l u e___ <_ s'? C s''/ nP // CfA <5"S,~f S/S ,'csO <A i'isc / // r*-, /n rTc_ </ * - 21 22 23 24 Source (Journal, Vol.f Number, Pages,Date) 12 b 1^ ' ^4/ /O/O /il---/"bo . T~tyZ : 61 62 33 32 trier Sumar 61 62 61 62 63 64 T Mutation I\'c*rurchk II (197G) l`J3- I .'id O KlM'vii*r/N<H,lfi,l I>1 J^inif HionxMhrjil l*r<-ss .It f -II ) Mm'i-mI Kt-port, Mli. iii.it**f*"jis nd ,1 Mj,i. *m..I A I l**-ri* |i)i iMi 'MA. if Ihhi**' mouM*. M ilmliri in Ihl- lurril ntuUln'nt IH..I . Hi 113111 It'J. > fr*i * nrl***r in .hi .Ndturalitl. Imiis Hi l)ii* hr* X `in .iiln- , III him *. (l.SK- .( .i(iii< m m.iUUff . , ,t tit '1 I l*.\ in il striM'lun1 u| >, |t l M iimi.iiu ,|| I>m |, 1**111/ ini: tin m>iiii\i`. Kn* l m I III- Hu mill ,M IluiMJII* . it. i m.mn| II Ml ||I* \ Mill! CYTOGENETIC ANALYSIS OS HUMAN ClIUOMOSOMEN AND ITS VALUE SOU THE ESTIMATION OS GENETIC RISK M. kuCekova I'ediatrie Department of rontftradnutc Medical / nth to tr, licnrtic l.iihiinilnry n( Institute n[ llytticne and Hpulcmioloxy, Prague, Srohurnea -IS (( SSI>) (Received Ki*ljruiiry `2nd, 197P) (Revision received July 20lh, 197G) Summary A short review of present-day contradictory opinions on the usefulness of human chromosomal analysis in the system of chemical mutagen testing is illustrated by examples of the results achieved hy both conventional and banding techniques. The results include exposures of human chromosomes to ECHH and TKPA in vitro, and to KCHH. vinyl chloride and imuran in vivo. Exposures of human lymphocytes in vitro to the chemical to he tested for mutagenicity are recommended as one of Ihe tesls to lx* included in the system of mutagenicity testing, parallel with all oilier tests on mammalian and submammalian levels. The testing of human chromosomes of people exposed to chemicals in vivo is considered essential. Introduction What is the actual plner of eytogenetie analysis of human chromosomes m the system of chemical mutagen testing? There an' a number of contradictory opinions ranging from a complete rejection of this method, particularly for routine testing, to its acceptance as a unique technique for the esiimation of genetic risk. The cytogenetic methods suitable for the analysis of human chromosomes are well established, widespread and not too expensive, so that they could be used for large scale routine population studies ;ls well as for systematic experiments in vitro. Nevertheless, il is frequently objected that the chromosomal aberrations detected represent only a part -- and maybe the less important one -- of all genetic changes induced by mutagens. Cytogenetic methods cannot, for example, delect point mutations. I Inwcvcr, the factors that induce chromosomal aberrations usually cause point mutations as well [15--18 J. JO Bo 0) o 00 ro o>1 T y i-w y 'IT T 1 lTT~ _I VtiJ-T -7 - v y fVM | H 1 2-1 Another objection argues that human i>< rii>hi r:il lvinuhoevics iixnl lor to start a pro.-if testing, in vitro and in vivo, can yield only :m average level of ehriunnsomc changes occurring in somatic cells whereas for future general inns il i> nmiv exposed to F.CI The experii important to control the chromosome changes lhal oeeur in gonad,it eells. e\ piimi tes |] The detection of chromosome changes in somatic cells could he valuable wd^u I from the aspect of carcinogenicity: many mutagen- are also carcinogens and (l-a/.iridmyl )ph i i there is a hypothetical relationship between chromosomal aberrations and the found that KC ii origin of tumours | 21. It can also he logically assumed that the chemical that o Its effectivnexx harmful to somatic cell chromosomes is also harmful to chromosomes in The next ste gonads, even though on a different level. Quite a serious criticism has also been raised against scoring chromosome changes in human eells exposed in vitro, ll is true lhal experimental conditions in vitro are different from those in living organisms, where most probably each chemical substance is subject to metabolic and biochemical changes. If the cells are exposed in vitro, these biochemical changes are absent so Hint we get a some undeteeb technique is us in our experin chromosomal conclusion that use banding U-< simplified but rather artificial situation. In an nl tempt in avoid this discrepancy, the method using pre-aetivation of the chemical in some biological system before the experimental application is under discussion | 20,\ .S ]. aberrations esca However, tlv breaks on indiv Another problem is that many chemicals are cell-singe specific in their ac significant mm tivity ( 1.1 1 1. Cytogenetic analysis of lymphocytes from exposed persons m in vitro in our n vivo may reveal their mutagenic effect provided that (>, stage cells me sensitive A newly sta i to the particular chemical. On the other hand, in experiments with human allowed us to lymphocytes exposed to chemicals in vitro, we can tesl the .sensitivity of cells in all stages of the cell cycle. exposure in viv fore they start R&S 108271 Our laboratory is interested in all these problems and that is why we have chromosomal a undertaken a series of mutagenic experiments with human lymphocytes using and we intend t different applications and cytogenetic techniques as well ax different This study is approaches to the problems. Our objective is to find out the real value <f cytogenetic analysis of human cells for the whole mutagenicity testmg system. of the Inslituu Moscow, \ e this pmspc < Material and methods A significant of occupation. A short-term cultivation (06--5S h) of peripheral human lymphocytes |!)] was used in all our cytogenetic studies. Slides were scored blindly after coding. For a detailed analysis, mentioned in the Oiseussinn, the trypsin-banding tech nique [3] was also used. The resuits were mathematically evaluated using the Xs tost. .-.Indy eontinu. For the pa.o by other wurki II). Most of il agreement with Since the pn consuming we < Results and discussion collect and ana Looking for a model chemical substance for our complex ey togcm-tii study we chose epichlorohydrin (K01111) (1-ehloro-2.3-epoxy-piopaiie), a lii liiiutional alkylating agent with potential mutagenic effect that until recently has not been sufficiently tested. There were two reasons for tin's choice. The first was that this chemical is used in our chemical industry as a solvent and as a basic compound for the production of epoxy resins, so that large groups of to a mutagen f< we chose nine known for its > cytogenetic an,, Table III. The : interesting that cells while m < workers arc exposed to il. The second reason was linked with our oppor1 unity tes used for chromosome *jt is inure ifctal valuable amyi-ns and "Ms and the mical that is mistimes in lirnintiMimc I conditions iltalily each i f I he cells it we yet a avoid (Ids e biuluyieal Mm llieir ac|iersuns in .re sensilive till human iiy nf cells y we have vies usiny different l^tllue of ^B'slein. icylcs | '.I | el eodmy. duty techiismy the lie s|udy hi-fune- reeen tly uice. The ill and as ;iimi|is of H irltiuilv T 12.'. to start a prospective cytoyenetic study nf worker populations uccupationallv exposed to KCI 111. The experiments started with an informative si udy i.inn'niel| With exposures of human lymphocytes in vilru. In these experiments the effect of KCI 111 was compared with the effect of another alkylatiny ayeiil TKI'A (Iris (l-a/.iridinyljphosphinc oxide) that is recoyni/ed as a s irony mulayeu. We have found that KCHU is a 4--5 times less effective nmtaycn that TKI'A |12| but its effectivncss is dose dependent. The next step in this study was provoked by the question whether there are some undetected chromosomal aberrations if only a eonveniion.il cytoyenetic technique is used. That is why we also used the trypsin baudmy lrehm<|iie |.'i| in our experiments and compared both results. Our comparative analysis of chromosomal aberrations caused by KCI III and TKI'A in vitro resulted m a conclusion that for larye cytoyenetic population studies it is not necessary to use bandiny techniques, U-cause only a m-yliyihle fraction of all chromosome aberrations escapesdetection by conventional techniques j v However, the bandiny technique enables us to detect the distribution of breaks on individual chromosomes and their parts. There was detected a hiyhlv siynificant non-randomness of break distribution caused hy KCI 111 and TKPA in vitro in our material | Id |.* I A newly started production of KCI 111 in one of our chemical plants has allowed us to initiate a prospective lony-tenn study of workers with KCHI! exposure in vivo. Peripheral blood samples taken from Lhese workers even be fore they started to work in the plant served as their own control. Another chromosomal analysis of blood samples was repeated after one year's exposure and we intend to repeat it 2 or 3 times after at least one year's interval. This study is beiny done in co-operation with the Laboratory of Mutayenesis of the Institute of Medical (Icneties of tin* .Academy of Medical Sciences in Moscow, where half of the all material is scored, 'I lu- first results of our part of this prospective study are shown in Table 1. A siynificant increase of aberrant cells was found in peripheral lymphocytes of occupationally exposed workers after one year's exposure to K<"HI|. The study continues and the final results will he published lati-r. For the past two years, the mutayenie activity of KCI III has also been tested by other workers in our institute usiny different mulayeninty tests | l*Jj (Table 11). Most of these results show the mutayenie effect of KCI 111 and arc in , ayreement with results of our cytoyenetic study in vivo. I Since the prospective type of study in men m vivo is laborious and time : eonsuminy we decided to try an opposite ami more common approach, i.e. to i collect and analyse blood samples from workers who had already been exposed I to a mutaycn for a lony time, at least for 10 years. For this informative study , we chose nine workers with lony-lcrm exposure to vinyl chloride, which is ; known for its carcmoyenic and mutayenie effects |-l,7,]-t|. I lie results of two ! cytoyenetic analyses conducted in each worker of the yroup are presented in ! Table 111. Tile time between the first and second analysis was 8 months. It was intercslmy that in some workers we detected a \ery hiyh increase of aberrant eells while in others the number of these cells remained at llu* control levels. N'on-homoyeneity of cytoyenetic results found m this yroup by the first and W: ftiVS ei.280i s*u --~~ -,"*~7>n nci TABLE I LONG TI RM STUDY OF WORKERS EXPOSED TO F.P1CIILOROU YDRIN Number o( crlli cored CcIIj u-ith aberration! Numbn t Tvpea **l aberration Number of chrometid break* Number of chromatid mchtnift Number of chromosomal break] Number of chromosomal CKChange] Worker* viih r.cun i i 3 573 3 336 41 67 1.2 2.0 * 31 34 \ s 7 21 4 7 1, Before rxpmurf, control}. 2. Alter *>nr year of ^xpnsurr. * Si(rtifictnU> higher than in rnntro| (/' *T 0.01 k Number of aberration* per 100 cclJi 1.2 2.0 Numbrr ! break* per 100 cell! 1.3 2.4 Number of t>P* 1J 1y i i I i i rr 5 < rr EL ? < ~ C. * C -- 3 r 's c ^ ~ " r- < s eg c c o m7D5 ' 'T 3~" ",--7r 3~r"- oTPj ~ ** 3 O. 5 .. 3 C 3 : c : t ? ^ r. c r 2 2 cr` C- X 7T ^ X- CK. 1.DcC.^3HiS< {OO/ri ' S"tT 5 <" * & =* 3^ '^33 c. S p. Li Vs 3 W " ^ 3" .i. s * g. S tu </> 3- 0 ^ 01 ' s E. 3 3 ^n c _ r K. O <n - * J i X3 ^^-a J j. 3- nk3. *--"- Ic 1H TABLE Jl TEST INC*. UK MUTAIIKNK* ACTIVITY Ol J-rilil (l!iT t I'lTM Ti**l I t'-slmi M -,l. in Tml Kit mii'tiinic.imsin | J *> | I i t-< I ^u\ | iy) Cyiojvitrlic n*ly>4? \*t bonp-nurmw ri-Jh of rrwmnul\ | I!> f U**l ||y| llumitn It ni|>hnf> ir* npinurp in \ur<> | 12! llwmn |\inphnr\U`, in vivo , MuU^'nic Aciivits, U. !)**! ({pprnilrtK'i*. Silloi Mli/I.l I \/ill I III >1' III >! ,\f//rmnWM l \ (thitnuriu m Moutc Clnni'-sp lirtinsi rt Mnust* I'fflphrf^l I vU|>Ihh'\Irs IVriphi'i.iJ I v mi iilww vl r\ M rsulu I) f) * |) t () *, * |) * 30 & U> o 00 ro also by lilt* second analysis was unusual in our material. The increase uf aberrant cells delected by the second analysis occurred in lymphocytes of some workers who showed no cytogenetic changes at the time of the first analysis, and there was no time-de|>endent effect'of exposure. We hypothetically ascribe this observation to an irregular exposure due the cyclic character of the produc tion process. The results of another cytogenetic study of persons exposed to chemicals in vivo demonstrate a dose-dependent and time-dependent increase of chromosomal aberrations in the chronically exposed persons. 'Phis time Lhc chromosomes of patients treated with cytostatic Imuran were analysed (Table IV). The upper part of this table reports four nephrotic patients treated with Imuraji for G months up to 2 years, the lower part of Table IV describes four patient^; with kidney transplantation lx-eause of glomerulonephritis, who wentreated for fi months up to 7 years. The literature data published on the effect of Imuran axe rather contradictory as to its mutagenic action on human chro mosomes in vivo [5--7,10). Our results seem to indicate that Imuran has a limedependent, and probably also dose-dependent, effect on human lymphocytes because all the patients treated with this drug for .`1 and more years without interruption showed a significantly increased number oT cells with aberrations. 'Hiesc findings can also explain the negative results found by some authors in patients treated with Imuran for a shorter period. Conclusions What is our opinion on the use of human cells exposed to chemicals in vitro and in vivo and its importance for the system of mutagenicity testing? Present cytogenetic experiences lent! us to the conclusion that experiments in vitro provide.a unique opportunity to detect directly the sensitivity of human cells to the chemical tested. Kspeeinlly dose dependence could he tested easily. We believe that these tests should be included if possible in the routine testing system parallel with all other tests on mammalian and sub-mammalian levels, and if so, the pre-activation of the chemical to he tested for mutagenicity should be preferred. The testing of chromosome changes in lymphocytes of persons already 9LZ801 SSH wifM --m 'ti m (m'imimin imxn --'tin li mini TAHLF III CHROMOSOMAL ANALYSIS OF WORKERS PROFESSION ALLY EXPOSED TO VINYL CHLORIDE Patient Number o 1 cells Krrd C*U* wilh iberrrlioni Number Tt Trpet of >bmi(ioni Number of of tViro* mat irl break t Number of rhr>* malid ex'* thaneet Number of chromoaomaJ breaks Number of chromoiomai ex change* Number of deipirali ra tion* per 100 cells E.H, 100 i 1 1.0 5 1 40 i 11 100 0 00 0 0 0 0 0 A.T. 1 00 *00 4 1 4.0 7 1.0 0 0 0 1 01 i 00 j.p. 100 4 4.0 4 0 000 100 2 2.0 0 0 1 1 0 4 i 4 2 F..J. 100 3 3.0 1 1 01 0 100 G 6.0 4 0 200 3 6 K.K. 100 2 2.0 2 o o' 0 0 100 7 7.0 6 I 000 3 7 r.M. loo 1 1.0 1 0 000 loo 2 20 1 0 i 00 t 2 A.It. ] no l0 1 i 1 .0 1 1.0 1 0 000 0 000 1 1r J.K. 100 i 1.0 t) 0 1 00 loo 4 4.0 3 0 1 00 I/ 4 M.M. 69 0 00 0 0 0 0 100 1 1o 0 0 0 1 0 0 i C*ntr*>Ji j:.:o 41 1.2 3 1 1 ~ 40 i-a i * High r xpmurr, mull' rhjn :>Of) j>.yi .*ii b r 3lNl p.p.m. Number of per 100 cell* Number Eipoaurc (year*! 11 0 3 1 4 3 i c, T a i <i i i .4 1) 2 13 4 0 1 0 0 0 0 2 2 2 l 0 U 0 i\ 2 1 0 14 10 (S S bl 10*8 months 9b 9*8 months 1G J 16 * 8 rnontht 12 J I 2 * H rn'ini hi li * 1 * 8 nmnlhi ii * 1 1 K nu>n:hs 14 1 1 4 * m.*n: hi Its ' 16 * A monihi 12 ' 1 2 t* n>rMht TA IILH IV j CHUDMilsiiMAI. M1ERR \TIONS AKTMl KWOSl'Il F. Ti KiHAN IN VIVO I r*< O-nln.K 357J i 41 in 1 .2 3 I * H.th r^pmurf, motr than 500 p.p.m. ^ ). tpfuufr 500 p.p.m. n \ tO 1 2 fi 1 months * 0 1.2 1.3 11 TABLJ-. |V C* IHOMOSOM \ L ABf il R ATI ON.S ATTK K EXhJSlRKTO INfUR AN IN VIVO Pitunt Ac (yrjfi) Years rpj treatment (itoic prr day) A.5. M.tJ. M M. J.M. L.O. M ti A.V, u..v AO 1 7 17 1*1 ia 42 22 26 27 0.5 (25 mi) 1 (25 m[| 1 (1 50 mi) 1.5 1100 mi) 2 (1D0 mil 0.5 (100 mO 3 (t- 5 nil) 3 \ 1 2 3 m (} 5 wars 200 m ( 2 vcjr 1 125 m| Number of cell* cured 200 200 200 200 200 200 200 200 l 53 Cells *Oh aberration* Number % 3 2 5 0 -1 K 912 * 14 ' 1.5 1.0 2.5 0 2.0 30 4,5 6.0 9.1 Type* of abrr rat inn Number 0/ ehromatid breaht Number of chromaud e ec h-n(r 20 00 40 00 40 20 5U 60 *) Number of chro mosomal breaVs Number *( chi mosumal e rhanics N'umWr of a.iptftli/aliuru Number of abrf* rations per 1 GO cells Nvmbrr of breaks per 100 C i lls Number of upt 0 1 0 1.5 2.0 0 2 0 0 1.0 t.o 1 * 0 0 2.5 2.5 0 0 ,0 0 0 0 0 0 0 0 2.0 2,0 1 3 1 O 3 0 3.5 4 2 4 0 55 75 1 4 b 0 K.O 1 l.J 0 3 4 3 i l .1 t 2,4 1 C*utcn| #f-U| ..I 20 l>< r v. <11% 10 1000 2i 1 .4 1'J 0 5 3 0 1.4 t fi 5 A Sijfii hr j nl iiu-ri'jiii1 (/' < 0 005), 1 9Z2801 SSH 1O^' I i;io exposed lo suspeeled mulauens in vivo s.-i-ms in In- ;i sul'l'iei.-nlly v-nsiiive method wlweh r;m he used ;is ;ui indirahe of human exposure. In nor npuiiiin, this testing melliod is essi-nii;il and should hi- used wh'-neu-r pnv.ihle. Muht <) Kl Aehnowled^emeiU The author is cniteful In u:i lor teelinie;i| ;i.ssist;mee. I'oli'vkov :i for . o opt-raliu.i and to Mrs. .1. Novo! . Keferenees I 11*` IM I*" I . M \ . II It, l>Mth .mil .1 N |ti I 11 >t < | . M* I III II ||.|*l I I. I i* * > *|I it .|||< ii.iIuim IMOiliH lp,,| (I) Chrmu .iK .iii.l it>m/ ihl hull.111*n, Mul.i I mu K i I *V ) i I 7 I i! ` *1 It . IMilui'i'il I'lifMmtisnm.il ,ihi-rr.Mimis m man, \t|. .m*'t m I In man i .* m-i a \'t*i. . I'l.-riuni IWi, N**tt Y.*fk lOTii. IM* .*1 Hu f k hi ill lor, (1. |) .mil U 1\. ('iidmii.s. 11. | In- ttii iik.i li.iMiliiu- ) i. 111 r 11 i>| i lit mimi m tint* ch.iiu*` tin rim4 I'mlirv (`Mii' i|.m i`|.i|imi-nt'* 1 i*l 1. (*fH II s , 7ft i 1 0 7 7) i'-M 7 I l 1 htrairn.tu. \ ,. I\ , I lirsi hlioMt .liltI .1-1 il I, iii> It lil**i uI* im|ii>miii- .ihi| r|iiMiiiiiMinii> .ilieir.111,,11^ Mtiiaicui k.-s.;h 11: t r) i.;i h;m. f> l-tu-rli*, I* , W. IliiMsti'io, h. I'rrmcs, UmiiiiHsuiiM's in pain nls in-atnl with limir.m, 1 |u m.mi-a-n.-Mk, t UlMiMMtil t;i Ii KriritrirU. (' ami \" `A'uihftl, (,lhmiiiisoriii>n,lliienmii.ili n Hml Ih-h.imlkmt-, nut lmur.ui (A/allm.* I'fiM) u " It N i't**tittanspl.iiii..I ! Hi*'in, 11mii.mi'i iielik, K < 111V< | if M'l if'l I , 7 tiiii't'(`f.niul"1 K,, 11. , .1. LiiuKmm, [, | Iih-mIm-i p. N.T. \,ii m.in nail s (kii,rni.m-( .lint, riifomoMimi' .i)n-rMium\ hi -t i* k*`fs *' t**s'41 to t iii% I* It!m uI.-, I,.nii-t i I ( | *i7 *41 I Vi H liiilim'i, 1' .1 < Ktm-itl, I*. IhKni lt amt It. ) |\i1h-i , I 'hi mints..mr\ m p.n |i-ti\\ in .ih ] v il A/ . lln*)ir im . I luMt.inci'M.'t ik , | K | 1 7 ;11 ii.'l I L'.'i'.. U 1 lu iifr liiril, I). \ . I rf-ti | in-v (*`v i-ulltin-.J limn small iimm ii l.i uIh.|.> ,nn| tin- pri-p.ii .il mu ( mul,pMa'a- i liinmosimies In In-alrm-Hl \s till lt\|*"toini M I. M.mi ..................1 tU i |*lli!i| .I.'IK 10 .Irnsf-n, M K.anil M, S^lmrt. t4lmnmsmM.-- .iln rr.ilimis hi Imum.iii r> II. (i.IImw Hit' tn*al iii',n i unit Imurjii. \i*U M**il. N#`.mil . 1 70 (1 `MMi > '.M!i AM) 11 Kihlimtn, II \.,.X Mtir(`l(i|, II. I t*r IJ* s atul K. Nilssmt, Mi* uili.iin run id |n < al Ii-iik- hT tl**` Ir* *|it*-u* t-irs >al rhiimiiiM'm.il i I h - rr.i (in Ms imltiri-O tp pl.irti .mil .ihimi.iI i -1 -11 \ |i\ iluHiti.il hhI hIivmi.iI a,:-nls. MiilJlu.it l(*>., io* (1 *17-11 |(:* \Z'Z tI il Ku^'io\,i, M , / I1. t|iv k i *x j. I(. Si .i m .m| V. M.ii i hi s'k , M ii I ,ii. ii it i | fi-i-1 < t ( * m<-U U nit \ ilr in t i-si mr tip Itti mi,hi 1% iiit>twM\Ji-s in vjtru in . i.iH|,iri\oit inih I I I* \, Mm..lion H.-, .11 |pi7h> 'J 7 I ,,'7s 1 ,'1 hniVriH.i, M <i it 11 7. I'olltkiit.i, H.hhIihi* |<-i|ihh|ih-s .mil Uh- l> I 1 * > ..I 4lii.un.ist.Hi.il i.i( u*iis iHiltit-t-iJ In i.Hltaliitu jiut Klktl.vtHiit >t I'.t'H t s 1M*.\ .uni pu *J7*I 'J*m In l r hi . \tti|,i (tiiii |(ev , .'11 | I * V*. i I Mimimt, l( . .1 M I'rtmi jhiI M.N', ./iiJiiim.h, I'i u| < i-m.i/ ui-h t.ifit i .nnimi1 v un < tiloruj.- \\>nkfi\t Litln-1, U lt07 lj :t`7 .T.IM. I \'lrh ii|\ W \V .. I i l Mil'n'In- l*-r!HHtH-*. m Mtnl.tt'.t in* Hi I. -t UK-, N i*.-,, \ s Mill NI I H >I1S , .1 `J < I * ( 7 .`l I Ki. ii*. 1 li Nil hills. \\ \\ . S | |`.lt | I H.llll`4- 1 I 1 .l| |l M| S ( 1 | It- 4- It I HIM IM Ml H ,1 I f I .4 I 11 t|l\ |o| HI l|l . I lit IIOII I ll-,l| I || iVrspi-rl.. i. ( p7:J \ 70 1 K.l, 17 Si'ltli-H'rm.H 1h- . I (107!) ton 1 ) I. Uihminuimi' .ilimi.dptni hi ................... .hhI m.i.isi s m \iii, Anli. 'I\t*il . 'Jk 1 M Nrjn * I I*t . 1. .ii h! (' Wil I. I*\m lx I it !*-, at hI Inn 11 .i 11 hiis . I It rmu * vt mu- t n .i 11 im-m I hi i i l ft. Itr 1 lu- iirtiltli-m .i ilit-iiiH'.il inn l.im-iH-Ms (`Ih-hiii-.i| Mm.ir.-iu-'ns It'Tn, pn 'J \'J Mi in M.mhim 4|s ami Man spimi'.et-Vi fl.if, I !> M.nn, l(.. M < Vma anil M, Km ' i-mi a . It** i*. ih-i ii i isk # *1 #-ji t( lili.r IiI mi ,n t l.i r t ! t i lu- ni.^iu *H,ti i-simisun-. Mtul /utilia 11 >t.111, 'i.ri ( I 07ii 1 I T 1 -1 hIf i!0 V'lV.fl. 1` atnl M, Vrli.i, |nnii4-m*r n| i iiiiir.imi'hrtm'o] mi tin- m rnumi Ir* <ti.< i v * I i t#i i .4H'imlu<<-4| fit ft Hit l IM Hill- Itli'.lk III I 11' L.I 4 lls. Mu I .11 H HI Ml'S , 1 ( l 'IlM | K7 1 Hut, I I I i t i 4 CM WIT PK.T! l)u i' UCci. (It.-. Sun- a su Will lytn in I; corn of t. of < ti<in ill i; vvivi Core W of < ()ro.' ne>'. ehilpoll lull. on :