Document nkZ8ypQozjNeOJLRv3LXpJKEG

. ... , f 1 L . . -. AmericanJ o u m l of Industrial Mcdldnc 7r447-456 (1985) @p hVenzene Hematotoxicityand Leukemogenesis \..Eugene P . 1 C r o n k l t s 1 / ~ . T ~ r e w ,T/.k~n,oue,jwor.rrd J.E{idll~, I niptit-to-twelve-wcck-old iiinle arid feninlc C57I31/6RNL inice were exposed lo air or hCIi7.CIlC viqwir in nir at a cotrcntrntion of 10, 25, 100, 300. or 400 ppm. Benzene at mtrclitrntlons of 100 ppnr or higher for 10 exposures of 6 hours pcr dny 5 days a week prtwlurcd a reduction in hone marrow ceJIuIarity and the nunihcr of pluriptcnt stem ccllr In the kine marrow. The fmctlon of stem cells in DNA synthesis was also incrcnscd. Expsure to 300 ppm 6 houun a dny 5 days a week for 2.4, 8, and 16 weeks prcxliicd n diminution in the rtem cell leveis in bone marrow which returned 10 those of aiiitrols 2 wcckr after hemme +xpo.wrc for 2 and 4 wcch, 16 week8 nfter exposurr for 8 weckn, nnd to 92% of controls 25 weckn after 16 weckn of c x p m m . Then was a niorc rnpid return of blood lymphocytes lo the control level. Mice exposed to 300 ppm for 6 hoursldny, 5 days pcr week for 16 weeks began dying at 330 dnys of age. whereas M dcntlin wtfc observed in sham-expod mice until 440 days of nge. The benztneexpind mice d i d in two waves: the first was from 330-390 days of age, with a second wwc cotittncnctng at 570 day^ of age. The firrt wave of martnlity WIM due primarily IO diyniic lyniphotiinta. The second wave wan due to a mixtun of ncmtliymic lymphomam ' auld BdId tumors. Key wmdri IrIlkcmqcncdr, hanopoldc stem CLUE, h e m e Icukrmngmd&hemopokllceffects lnfantc et al 119771 and Rinsky et al 11981) reported an increased incidence of GD M C & ~ R C R & W ACenrb Bmokhvm N a p Ld&tum i w n t h o l o g y , Yokohama Clty nivernhy, Japan CT.1.) ,.,,..:.Z.B+ '. $411 Cmiiklte et PI - Icukemin in Individuals wlio apparently were exposed to a timc-weighted avetage of OIIIY 10 p p i . Further analyRLs of the concentration of benzene in the workplace rliowd tluit at tlnits individual6 could have been exposed to as high as 300ppm. It is of niajor Itiilnirtnnce to dctcrmine if benzene concentrations at levels thnt produce no pticylopctrla or perturbanct of the hcmatopolctlc stem cell pol will be leukemoBettic. If tlih wctc tire case, it would ~ ~ p pthret idea of a no-threshold linear dose reqntnst hctwctn benzene and acute mydogenow leukemia. 'Ihc rtpincd obsctvatlona to drita clearly indicate that furtlier epidemiological studies and appropriate anirrid Ktudicr itre tsscntinl to establish the dosc-effectrelationship bctwcen exposure lo bcnzcnc, tlcprcnsion in I.reniatopoiesiu,and tlic incidence of leukernin. Accordingly, we hnvc studied the tffcct of vnriuus cniicentratiotlsof benzene on mice king exposed 6 houra/diiy for 5 dnydweek for 2 wcekR to 16 wccks. Olrwcrvnliorls include blood counts, lotal cdlrilurity ofhone iiiwwv, iniiiihcrs of pltiri1x)tcal stcili cclls in bone mrrow, coloriy fontintiim unit (CFLJ), friictioir of tlic stctii cclls in DNA synthesis, ilic iacidaicc of Icukciiiin, ntid the dcvclopmalt of solid tumors aficr ccssation of CX~W'S~"C. MATERIALS AND METHODS Eip~it-to-(wcIve-weck~Idntnlc and feninle c57B1/6 mice were exposed lo ccmiitioircd air or air containing 10, 25, 100, 300, or 400 ppm knzcne in chambers similar 10 tlwsc rlcscribcd by Hiniicrn ct a1 [1968], an isolntion system dcscflbed by Lnakln ct a1 I19701, ot in ~ I I I S R"picklc-jnr" dmttibers like diose dcscrihed by Leach 11965). Mice wcrc allowed frwdand wntcr ad llbitum except during exposun periods. Exlwmuren wet%6 liours/dny, 5 ccmccutive days/weck for 2to 16 weeks. In a scpnmte scrics of axycriiiicnts, ngc-nintched mice of the same strain wcn: exposed io normal air or 3W ppnitrcilzctic 6 tioursldny, 3 consecutive dnys/wetk for a total of8 weeks. At Ynrioui llriici during and ancr the exposure periods, five-to-ten riiice were reniovcd from both the eontrol and.lxnt~nc-exyoacdgroups, ancsthdizcd by cthcr, bled by cnrdinc pnirrture via mil vein, nnd killed by cervical dislmnrion. Blood UI~IIIIS,dllf~mmtlcilr;,tie~nntcmitq,and msnya of CFU w e n pcdomed 18-22 hours after tcnniilritiwt of tlic expisure p c r i d or alter a designated recovery pcricd. White b i d =I1 coudr niid i d blood ccH counts wctc ptfornied on the Mood qxcimens usin8 a Cttultcr clcaroidc counter. A hind leg wus rciiloved from a c h aniriial and the bone iiwrrriw W ~ iElunliad from tidier the fctiiut or the tibia and femur. In SOIIIC cases thc niarrow wns j'cxtlcd, while in other studies inice were assayed individually, Total marrow cellularity was dclerniined. Aliquots of some bone marrow suspensions were subjcctcd ta high qxcific activity, tritiated Uiyiiridinc cytocide, in a nidification of the method dcscribcd by Btcktr u a1 11965). This procedure provides the percentage of stem ccils in DNA synthesis. Briefly, cells (1.5-2.0 x 106/ml)w e n incubated for 30 minutes at 37OC with 20 pCilmmole 'HTdR at a concentration of 25 pCilinl(0.3 pglml Bymidine) or in a nontritialcd thymidine solution of a similar wnccntration. M e r incubation the cclls were washed In ice-cold buff' containing excess thymidine (30 pglml) and resusprided in thymidine-free tissue culture medium. Fmr each suspension (Intth cytcwida and control for cxposcd and shnm-exposed groups), rccip- knt mice wcrc fntnlly lrrndiatcd (730or 750 R ~ d s2, 50 kvp X rays) 2-5 hours before bcing given 4 X lo' nurlcnrcd hme niarrow cclla lntravenously to a m y for the CFU content by die Xll and MrCulloch (19611 procedure. 'Iliese recipients were killed 10 e --.. i I rernge of 'orkpincc 'prn.It in Idurn flo cukcniocar dose obscrvairopriate 'XpoSUre irdingfy, cxposcd e blwi in bonc ntticsis, ition of I I 8P )sed to amhcn by Leach Eriods. :parite iornral VeCkS. : wcrc ether, BIcmd hours White imcns nd thre cases Total were on of ntage d for i(0.3 ition. idine each Rip :fore CFU d 10 I ! Benzene bukcmcwnais 449 dnyr alter li\jcr.ti(m. Splccna were rciiiuvd and fixed In Douin's solution and the surfnoec coloiiies were counted. RESULTS FiRurC 1 RIiows the effect of inhaling 0, 10,25, or 100 pprn of bcntcne, 6 hours/ dny, 5 dnys/wcck, for 2 weeks (10 expsurcs) on the total number of nucleated cells obtnind fnm a feniur (mnrrow cellularjty) as m m r c d -20 hours after the final exposure. Ihcre was no effect of Inhnlntion of 10 and 25 pprn, but there was a sigriificnnt (p < 0.003, Studcnt's t-test) depression in the marrow cellularity by inliulntloii of lo() ppiii for 10 dnys over a 2-week intervnl. Figure 2 nhowa the effcct of lnllnling 10, 25, 100, or 400 pprii of benzene, 6 Iiourdchy, 5 tliiys/wcck, for a total of 10 exposures over 12 tfnys upon the CFU level in the fcrirur cxprcsscd ns pcrccrit of tlie ngc-riintclicd controls. Tcn nnd 25 pprn had no cffcct. Wicre wns a kignificnnt depression at 100 (p < O.OO1) nnd a greater dcprcssiicin irtlcr inhnling 400 pprn (p < O.ooo4). Piwurc 3 R ~ U W Sthe effect of Inhuling 10, 25, 100, or 400 ppni of benzene for 6 hourddiiy, S diiys/week, for 2 consecutive weeks upon the frnction of hemopoietic stciri cclln (CPU) dint nre in DNA synthc&i. Ilic fraction in DNA fiynthesis was less tliiin corlrol-icvcl alicr inhnling 10 pprn, but It wan determincd only once. There was nu cflecl alhr Inlinling 25 ppiii, but here was a slight i n m s e in thc frnction in DNA syntlrcsir n l h lnhnling 100 ppin (p < 0.17) or 400 pprn (p < 0.08). Thest cbanges arc of qllcat~oniibksignificance. Previously, when 400 ppin was given over a longer time. n Rh-JliifiC!anl increase in fraction in D N A synthesis was obscrved [Cronkite d al, 19821. -24.- P - -0 I 22 L 3a W LL 2 0 a W P 2 le- wd 0 i 3 10 2 5 100 DOSE lopml -FIR. 1. 'the effect of Inhalatlon of bcnzcnc 6 howl pr day, 5 days per weck, for a toml of 10e x p o s u ~ u p n the II~IIOW allularhy (total nuclutd calh collectd from one femur per donur). Mean f S.E.M. rhown. n 9 to IO per &ne p u p . .? '... ..a. i*. 450 Cnmkltctt al ,,IPD 3 J. 9 J. I O 0 a0 ca! BO LL 0 60 Y .4 a W 40 (L p: 2 eo 3 u(L IO e8 100 400 DO9E (ppmt R p . 2. 'flir rlliu.1tin CFU rnnient id inheling 0, 10, 25, IO,or 400 ppm knrrne 6 hwrn per day for 5 clryr inwrk lni a ltnd of 10 rxpmiirmover 12 &yr. Mcan f S.E.M. rlmwn. All dam m a l i z e d tn nmtiol voliie fiw each army. ?%rea repnrnte srrayr performed at 25,'snd JUO ppm. !wo assays a1400 ppm, d J rlnpla army at 10 ppm. -50 40 We 0E 30 V zfl 20 4 W z IO c 0 I O 23 100 4 0 0 DOSE (purnl Fig. 3. 7 k effect of inlulins 10.2, 100, or 400 ppm of bentcne for 6 hn per day, 5 days per week for I; rad of IO e s p s r n upon the fnalnn of ~ i t mctllr (CFU)In DNA rymheda expressed as petccm cytoclrlc by trldntnl tlrysildlm. M a n and S.E.M. ahown. ' I h f a~rnayr each at 25 and 100 ppm, two at 400p~ma, d una W D ~at 10 pm. I -ne Lcukemogencsls 451 Ancr inhaling 10,25, 100, or 400 ppm ofbenzene 6 houdday, 5 days/weck, for ten e x p ~ u r e sover a 12-day interval, there was no significant effcct upon the hcrnntocrit at 10 or 25 ppm, but a significant diminution in the hematocrjt was observed after exposure to 100 and a greater effectafter exposure to 400 pprn (data not sliown). The diminutlon can be explained in part on the basis of suppression in red ccll prduction but probably also iiidicates hemolysis because sections of spleens of mice killed or dying during exposure pcrjoris were found to contain considerable dcposits of heitiosidcrin. Figure 4 RIWWS the effect of inhaintion of 10,25, 100, or 400 ppm of benzene 6 hoursklay, 5 dnye/wcek, for 10 exposures over a 124lny period upon lymphocytes niitl grniiiilocytcn. TIicrc wns essentially no effect at 10 ppm, but thcreaftcr a doseeffcct rclnlioiidilp an the diiiiiiiution of lyniplitxyte concentration in the periphernl b l t ~ W~ l~ nRlq)arcnt. No effcct on the grntiulocytts was scen throughout the exposure Ixriid. Figlira 5 W I I ~ WtlicRlevel of CFU (percent of the age-iiintclicd controls) as a functlun of tliiia after exposure to 3W ppm for 2,4, 8, or 16 wccks. After 2 or 4 wccks of exlwisure, the CFU had returned ascntinlly to the level of the age-matched cotitrola 2 to 4 weeks after the last exposure. After 8 weeks of exposure with the first 1 for aaanys bchg 4 weeks after the last exposure, the CFU was appmximntcly 50%ofthe ired oge-tiintclial coatrols. There was recovery at 8 weeks, and by 16 wtcks after the last 400 cxIw)stire, the CFU level exctcclcd thnt of the age-matclied amtrols. In the case of 16 wcekr of exIx)twre, the mutts sliown arc the incans of two scpnmte expcrimcnts. The first many wne 3 dnys aftcr the last exposure, at which time the CFU level was appmxiiiiiilcly 27%that of the age-rnntchcd controls; however, 16 weeks after exposure the CFU level was still 60% of the age-matched control mice. In a single assay at 25 w e e k nncr exposure the CFU level was 92% of control. Pigitre 6 illustrates the effect on blwd lymphocytes of inhaling 300 pprn of benzcnc 6 houm/day, 5 days/week, for 2,4, 8, or 16 weeks. Thc top panel shows the 0 LYMPHOCYTES ai ORANULDCYTES '! 1t i 452 Cmddtaet.1 I20 CIU - RR Lro AFRR B E N ~ N E~XPOSURE t 300 p p 8)Wita.%idb 0 was LXPOSURE 4 WKS CXCOSURL 0 r, v) 4 0 IS was EXPOSURE = 20 luh . M A N Of 2 CXPTS ', ----44 2 4 6 I 10 I2 14 I6 25 T I M E ( W K S I A r f f l l L A S T EXPOSURE VIP. 5, Ilir CPU levels (erprcnaed an pcrccnt cif ape-matched conrrnla) In mlcc thnt were expstd to 3lW) ppin, h Iiortrn pcr dny, 5 dry1 pcr w n k for2,4. 8. or 16 weekr and then observed until levels had rtttirimed to niwrml. effccln of 2 weeks of cxposure, counts first being made 2 weeks ancr termination of expwnire. At 4, 6, or I6 wecki poitexpsurc~thm was esscntinily no difference bctwccn tlic ngc-mntdied controla nnd thc bcnztnc-exposod mice. When mice were c x p c d far 4 weeks to 300 ppm, with the first counts k i n g 2 weeks after the last exposure (smmd panel from the top)*there wa5 a significant difference between the benzcnc-exposed, and age-mntched controls 2 and 4 weeks postexposurc. At 8 and 16 wccks, this diffcrcnct was no longer evident. Afttr 8 wech of ntposurc to benzene, the first counts wcrc also made 2 wecks after the last exposure, at which time there was a significant difference (p < 0.002). By 8 and 16 weeks, there was no significant difference between the exposed mice and heir age-matched controls. After 16 weeb of expsure, thc first observations were ma& 3days postexposure. At this time then was a marked dllTcrmn bctw& the btnzure-expscd and h e age-matched control mice (p < O.UOR). Fur 8 weeks thercann; then was a v g m s i v e increme in the lymphocytc levels in the blood of the bentcne-exposed mice and at 8 and 16 weeks exposure there WRS no significant difference. Fj;ipcure7 Rhows the cumulative murtality'todate after micewere expstd to 300 ppm 6 houndday, 5 dnydwcck for a total of 16 weeks mmpnrcd to sham-exposed mice. Bcnzenc nninlnls began dying at about 330 days ofage, whereas no duiths were obsmd in the 8haincxpstd mice until 440 dnys of age. Froin 330 to 390 days tlrerc wnr a pnqmwive incrcase in the cuniuintivc mollality, The death-rate remained low until arouid 570 days of age, when a s t c o d wave of deaths commenced. Eight of the first tcn tiilcc that d i d had diffuse lymphonma with thymic involvement. A mixture uf lymplomsta and solid tumors, primarily of the zymbnl gland and ovaries, were f t n r d in niico dying during the later pcdod. Table I lists tumor incidence by h ~ h g k tdy l h & DISCUSSION Our studies clearly show that 16 weeks of exposure to 300 ppm benzene, 6 hourdday, 5 Jaydweck, products a major diminution In the hemopoietic stem cells I I Ir Benzene Leukemogenais 0 CONTROL r0 TREATED AFTER 2 W K S EXPOSURE 453 Ised to hhad AFTER 4 W K S EXPOSURE In of `ence ! AFTER 8 WKS EXPOSURE were : last 1 the id I 6 f y 1me, AFTER te WRS EXPOSURE here I cant 24 8 16 TIME AFTER LAST EXPOSURE IWKS) XkS here Rg. 6. The effeci nn b i d lymphncyten of lnhnling 300 ppm of bavme 6 h n per hy,5 per itrol week fur 2, 4.8, a d 16 weckr a d the regemmion r b r turninationat u p r e . the xks with iiicoiiiplcla recovery 16 weeks after termination of u p s u r c . Wlicther recovery is ever cc)nil)letc is not known. In contrast to the CFU, lymphocyte levels in the pcriplierni t h x d rcturn to normal levels. As reported previously 1Cronkite et al, 19R41, In tlic aiiiinals exposed for 16 weeks, there is a very significant increase in the jncirlnicc of Iaikeinia Uint can bc correlnted w i h major destruction ond incomplete regenerntion of the hemopoietic stem cell poolrr. Whether leuktniia will appear in mica in wiilcli Iliere is no dlmitiutlon in die hemopoietic stem cell pool remains to be wen. Olhcrn tinve reported efftcur of benzene MI murine pluripotent stem cells. Gill et ai 1 I V W J fouiid tlmt cxpo~ureto 4 . 0 ppm benzene 6 houdday, 5 dayslweek, rcdiiccti 1110 CFU in bone marrow cells from 8.8 to 2.2 per 50,ooO cells after 6weeks of cxpoxuro. In contrast to our rrtudics, they did not observe a decrease in bone marmw ceilulnrity and did not cnicuiate the total number of CFU per femur. Gill ct ai I ICrRol r q m d that 2 days of continuous exposure to SO0 or 1,000 ppm resulted in a decrease in nbxolute marrow cellularity by 2 days, comparable to the dtcruise we obscrvd nftcr iiiterniitrcnt cxprure. 6 Oreen et ai [ 1981J expc~cdmice to gradui doses of benzene (1.1 to 4862 ppm) ils for 6 hours pcr day for 5 days and measured the CFU content of splccn and femur 3 I 1 I .. . . -4 0 1,. - b MORTALITY ANtl k U K EMIA 1NCIDENCE AFTER BENZENE EXPOSURE III IIIi 0 0 SHAM EXPOSED * e BENZENE EXPOBED f5OOppm, Bhrr/d, Fig. 7. Cumitlnllve mnilnllly aner cxytmm of mlce to benzene 300 ppm for 6 h r n per day. 5 day8 per weck Tor 16 wrrk8. ronipnml ta n h . m c a p d mlce. The control population at rink totaled 88 mice a d Lhe exposed population LIY niics. TABLE I. Tuntnr Ineldmrc Amnng Sham and ncnzenr-Exposcd CS7 Female MI& ECTcctive No. rt rink Shsmcxpsed 88 (loo)' Benzene-Exposed 89 (100) Leukernla (all t y p ) LymphorVtk nenplnm (thynifc) LympIwcytIc neclylnnm (nontIiymic)b Myelogamw lwkcmlr b k a n L (IyPC na( 8pedfid) ZymW Ovarian nwnor HC~IO~ 0th- ncoplum N o n w i c iakrnd Surviving 8 (9.1) 1 (1.1) 2 (2.3) 3 (3.4) 2 (2.3) I(1.1) 0 (0.0) l(1.1) 2 4.3) 4 (4.5) 65 (73.9) 20 (22.3 10 (11.2) 6 (6.1) 0 (0.0) 4 (43) 16 (18.0) 8 (9.0) l(1.1) 4 (4.5) 6 (6.7) 41 (46.1) ~ *ABof June I f , 19M.'Ihlnl liinitn exceed total number with tumorduetomultiplelumorhcidcnce. 'Number and puremrp nfI-rlrk trcylutnilon the Iaiter rhown Inpmnlhuer. hlduIymFhocy(lc Intkcaiila a d d d lymphoma& qncldea bcnien a d nuilgnantrplilcnnnldtumor and lymphnzpkhdom. 'Mice dad without neuplrrmbur ww1inflammation, hemopoietic dl-u, dc. hours after Uic ~ASIcxpcmrc. At concentrationsof bcntene > 100ppm, hey observed a significant duction in CFU content. Studies on recovery were not performed. Irons et a( [lW9J did not rtutly ~ t cmells directly; they did, however, examine die DNA content of the bone riinrrow ctlls and the incopration of tritiated thymidine. Thcir studies 8hmvcd a sigirificclnt increase in cells in DNA synthesis, appearance of tetraploid cells, a d a rductlon in fhc spedfic activity of tritiated thymidine in DNA, suggesting an abcrmtion In lfic cell cycle. Uyeki et ai (19771exposed mice 8 hours/day to 4,680 ppm benzene for I-, 3-, 3.5- and 8-hour =dons. Ibcy found raiuctions in the CFU and CFU content of bone marrow, but only nltnsurcd the icvcis 24 hours a n u the last cxposurc, compared to c al, . hair bcn; ieuk AKI diffe c57: Thei At tf exPo expo high1 conti aiter 420 c expo! signii signii after incidt obser incide misle; schcdi regent 1 penurl thresh4 there r REFEI Aksoy h Aksoy h ot Aksoy Jv P Aksoy M 44 Bccker A al 301 cronkirc OD[ mnkitc E 5 dsyt ;8mice Ved led. the ine. :of I& 3-, of red I -I - Benzene Leukemqenrsb 455 to our long-tern1 studies of many wteh and our previous 65-day study ICronkitc et al, IUSZJ. Stoiicr el iil [ lVEO] exposed flRSIJ mice of two genotypes-die leukemia-prone hairless iiiirw (Iir/hr) and the haired mice (hr/+) resistant to leukciiiia to 400 ppm bcni.cnc for 6 months commencing at 7 montha of age. No effect on the incidence of Ieiikciiiin In cltlicr the (hdhr) or (hr/+) mobserved. Snyder et a1 119801 exposed AKH inice lo I(I0 ppm for 5 dnye per week for a lifetime. Then was no significant dirfcrcnc! in Inridenct of lyinplionia in contrnlr or exposed mice. They also exposed C57B1/6Jiiuilc mice to benzene 300 ppin 6 houdday, 5 dnys/weck for a lifetime. Their ohrtcrvnlioins on lymphoma induction ceased after ahout 500 dnys of exposure. A1 this titlie llicre was n rignincnntly grcnter incidence of lyiiiphonia in the bcnzenc- cxpscd iiilce nR coiiipnred lo flie miitn)ls. Our-fitudieson C57Ill/W fcmale mice exlw~scdto the ~iiiiiecoircnlrnlion 6 hourddny, 5 dnydweck for 16 wccks induced a liiglily sigiilflcniit incrcnsc in llic Iiicidcncc of lcukemin [Crorikifee! AI,19841. Our coiifiiiuiiig mdics showcd a first wnve of lyiiiplioiiia conitiicncing at about 150 days nfkr cxposere. A sccond wnve of lynipfionia and solid tumors cornincnced around 420 dnyR nncr cxpsure. The present report shows the incidence up to 580 dnys after exptmre. 'Ilil~exposure rcgimcn nlso d u c e d the hemopoietic stem cell population tiigiiilicnntly; Our studica sirow tlint txpwurc for 16 wedlII to 300 ppm TCSII~~iSn a si~iiificniillytllffercntpntleni of lymphoma and solid tumon than has been observed alicr lifctiiiie cxlmum. This suggests thnt continuous exposuretithcr suppressesthe incitlcnceof lyiiiphomn or eliortcns the l i f u p n of the mice, so lymphoinas cannot be observed. 'Iliis phenomenon may be similar to radiation cnrcinogencsis, where the iricidciice ~ I E R tlitough a maximum BS the radiation dose la incnascd. Our sltidlcs suggest thnt lifetiirie or very proiongd cxposurc studies may be niislcndiiig. In clicmicnl leukcmogcncsis it appcars desirable first to determine a dose sctruliilc I l i d algiiificantly dccm.scn the hemopoietic stem cell p o l without complete regcncration ntd dien to deterniim if such a regimen is leukemogcnic. I1 In of innjor i m p m n c e to determine if an exposure rcginicn that docs not pcrlrirfi lieiitqwlais is 1eukciiic)piic. If 1t la not, we would fiuspcct tlint then is a thrcaliold cmcailration. if R U C a~ regimen retilains lwkei~iogciiic,it would imply that Uicn imy rwt bo nUrmdiold. 1 REFERENCES I Akrciy M (IWki): Mnlignancy due inoctupeiinnal exprnure to benzene. fiemacolgica 69370. Atmy M (IVHIL):!)Iflmnt l y p n of tnnll~nancladue to omrpaiional expure to k n e . A review of reccnl ttt*mnlnnr in Turkey. Envim R a 23:181. Akuiy M, M c m 3 (I97R): hilow-up ntudy on the monallty and the developmcnt of lcukcmia in 44 pncy!cqmic ptientn 4 t h chrmic e x p n u n io benzene. Blood 52:ZES. AkMy h*fidem S,D i d 0 (1974): teukemfr in lhoa worken expned chronically lo bcnzene. Blood 44:RJb. Bcckcr hJ, MrCulLtci~EA. Shnlnnviicb L,Tlrr J 8 (1965): The effect of dlffdn8 demands for blood cell prtrluctkm on DNA rynthuir by hemnpoidc colony-forming a l l 8 of mice. Blood 26:296- 308. Cmnklte El', lmnie I, Clrntcn AL, Miller ME. Bulllr JE. D m RT (1982): Effecu of bmztneinhrlPtion nn h e nlurinc pluripotent Itern cell. J Toximl Envlr Health 900:411421. Cronklte EP, Rullin JE, lnoue T, Drew RT (1984): B t n a i~nhalation produca lcukrmir in m i a . IbxiCd Awl M # M ~75:358-361. 0111DL).Jentinn VJ, Kempen RE, Elllr S (1980): The lmponance of pluripolentiai ucm cells in W e (cniclly. IbxIcobay 16:163-171.