Document n9xmYvdbvbaDoakKX1aOww4N1

? 0 0 0 N W , Chemical Leukemogenesis: Benzene as a Model c Eugene P. Cronkite BENZENE CARCINOGENESIS IN MAN BEYI ZENE has bcen acccptcd as an cnvironmcntal carcinogen by US regulatory agcn- cics. It is ubiquitous. being produced by natural processes in nature and in hugc amounts in industry. For cxamplc. in thc US. 1.6 x 10' gallons werc produced in 1980 in thc pctroleum industry alone." In 1897, LeNoir" described a a s c of purpura in man. and in 1928. acute lcukcrnia in a person cxpowd to benzene was rcportcd by Dciotc and Borgomano.I7 Thc cvidcncc of bcnzcnc being a human carcinogcn is based to a Iargc cxtcnt upon thc clinictll studics of Aksoy et d'-'and Vigliani and Saita." Thc clinical cvidcncc incriminating bcnzcnc is dcscribcd by Aksoy ct ai.' Thcir study is summarizcd. I n Turkcy, chronic bcnzcnc poisoning occurrcd in workcrs involved in thc manufacture of shoes and slippers whcrc bcnzcnc is used as a solvcnt. Of 34 individuals with pancytopcnia, six subsequcntly dcvclopcd acutc lcukcmia. Aksoy statcd. "from 1967 to Scptcmbcr 1973. 26 paticnts with acute lcukcrnicl or prclcukcmia .wcrc sccn in Istanbul among 78.500 shoc workers cxposcd c1ironic;itly to bcnzcnc. The incidcncc of lcukcmia ainong thcsc workcrs w;ts 13/ 100.000. which is a marked and statistically significant incrcasc ovcr that for lcukcrnia in the gcncral population and provides cogcnt cvidcncc for t hc clinical iniprcssion of a lcukcmogcnic clTect of bcnzcnc in man." Aksoy,' in his most rcccnt publication. rcportcd that 1cukcmi;i in bcnzcnccxposcd workers dccrcascd a h the phasc out of bcnzcnc in Istanbul. and thcrc was no lcukcmia obscrvcd in workcrs during thc last 3 ycars of his last study. implicating bcnzcnc as a probablc causative agCn1 in the earlicr intcrvais. That benzcnc perturbed hematopoiesis has long bcen kno\\n. Scilinghoand Wciskotten et 31' in 1916 clearly demonstrated that injection of benzene produced pancytopenia in rabbits and other mammals. Evidence that bcnzene might be Iru- kernogcnic was first deduccd from the sequence of evcnts in human beings. I t appears that aftcr intermittent chronic exposure to inhaled benzene of the order of 200 to 300 ppm for unknown' periods of time. pancytopenia may develop aftcr a variable latent pcriod. In those individuals th;it survive after variable latent pcriods. acute Icuhcmill may dcvclop. The leukemias dcscribcd haw bcen ;icutc myclogcncous Icukcniiti. Iymphob!:istic lcukcmia. and erythroblastic Iculemia. Whcrcas thc incidcncc of pancytopcni:i in workers cxposcd chronically to bcnzcnc is unqucstioned. whether thc incidcncc of leukcmi3 is significantly incrcased has bccn questioned and will be considered next. Ott ct 31'' studied 594 occuptltionally cxposcd workers. Tlitcc cascs ol' lcukcrniii were obscrved when 0.8 was espcctcd. Thcir statistical analysis showed P 2: .017.The! concludcd. "work histories and lack of nicdicil history made a rctrospcctivc ;isscssnicnt of ihc possible relationship to bcnzcnc csposurc vcr! j ud gmentaI. '* Furthcr incrimination of bcnzcnc 3s n human lcukcmogcn has bccn suggcstcd by the dct;iilcd epidcmiological studics of lnftrnte ct al.j4 Rinsky ct Whitc ct ai.'* and Infante and N'hitc.!' In thc first place. 'thcrc arc a large number oF uncertaintics in thc estimation of huni;rn cancer risk bccausc many of thc important conditions with which onc is conccrncd lie outside the rangr: of scicntilic obscrvation. Gaifcy." in criticisin of Whitc et a1.72points out potential defect in risk assessments. namcly. thc age-spccilic intidcncc of cancer varies with agc and is incrc.:lsins in most instiinccs. The assumption tliiii rcI;iiivs risk is constant for :ill agc groups is qur.stion:\bic. and usually lilsc. In response. lntintc et ;iIJ' point out that in rcality. they uscd I W O cumuI:ltivc mortalities for IcuLcmia; one for psoplc cxposcd ti) bcnzcnc and onc for thc !thitc malc popu1;ition aged 20 to X4 )cars. The ratio 01'ihcsc two x 100 is the standardizcd niorinlit!. rate (SAIK). It appears that t h ~ G' sITsy criiicisrn i> BENZENE LEUKEMOGENESIS - 3. not pertinent to the Infilntc et al" study, but is an important consideration in epidemiologicstudies. Van Raalte et a169 observcd that thcre is inadcq u i t e information on exposure of groups in which leukcmia was present and he also questioned linearity of responsc with cxposurc. The cpcrmissible exposure or time-weightcd average (TWA) imposed by the Occupational Safcty and Health Administration is IO ppm bcnzcne aver- aged over an eight-hour work day. All cpidcmiologic estimates of the incidence of any disease in a population chronically exposed to benzcnc must' be corrclated with the exposurc level. Infante ;ind Whitc" have shown that in the period I937to 1940,the TWA was of I50ppm in inspired air. and irom 1941 to 1946, it was 100 ppm; in the interval, 1948 to 1956.the TWA was 35 ppiii. from 1957 to 1962, it was 25 ppm. from 1963 to 1968. it was 25 ppm, and from 1969 to 1975. i t was 10 ppm. Certninlg the cxposurc of workers has been stctldily decreasing and it is wrll-documcnted in thc study of Scott." Of 3S.000 data points. 87% reprcsentcd cxposurc to I .O ppm or less and 98.4% were lcss than I O ppm. It is of interest. and perhaps explains why there havc been no reports of alleged benzenc-induced leukemia among workers commencing initial exposure sincc 1977. On the other.hand. further prospectiveand retrospectivc cpidcmiolosic studies on thc rclationship of benzene exposurc in the workplncc and i\ possible incidencc of leukcmia and other cancers are clcarly needcd. It appcars to this author that the evidence Tor bcnzcnc leukcmogenesis in man is ncarly incontrovcrtible. The crucial questions are as follows: ( 1 ) Is thcre a threshold exposurc of no etTcct'?(2) What is the dose-cffcct relationship? tnfantc and \V hi ell-:Y and \Yhitc et al" havc :iddrcsscd thcsc issues. Considering thc interval exposure of 1952 to 1974, Infiintc and White'? concludcd t h a t 42 to IS5 ppin p a r s is iiss0ci:itcd with :i 3.75-fold relative risk d Icukcmia. Thcsc investigators and thc International Agcncg for Research in Cancer" adopted thc onc-hit linear modcl. which assunics no threshold and that cvcry increment in dosc of benzene is accompanicd by :in incrcment in !he excess c;inccr risk. lnlicrcnt in the onc-hit hvpothssis is the assumption that ilicrc is ;i sinpic, uniquc chcmical cvcnt that is irrcvcrsiblc or that il`this evsnt takes placc on DNA.thcrc is nc, rqxiir of thc injury. Covitlcnt binding ol` `benzeneand its mctaboliies to DNA in vitro has been demonstrated by Rushmore ct al." Othcr adducts are repairud by known biochemical . mcchanisms and thcrc is no n priori reason why the adducts formed by brnzenc mctabolites cannot, in principlc at least. be partly rcpaircd. Chandler" has addressed in more detail the undesirable nature of the one-hit modcl. The problcm of chemical carcinogenesis, relationship to cxposurc dose, and conccntration of DNA adducts has becn carefully inalyzed by Hoc1 et al." They point out that the relation between the applicd dose and incidence of cancer is frequently nonlinear, but the tumor response is in some instances linear with the resulting concentration ofDSA adducts in the target organ. The most likcly explanation lics in the fact that many chcmicals are noncarcinogenic in t hemselvcs. but are converted to cnrciiiogenic mctabolites. The ph:irmacokinctics of conversion, dcgradation. cowlent bindins of ilCIivc metabolites. excision of adducts. and DNA rcpair will probably result in nonlincar proccsscs producing thc ultimate D N A adducts prcsumcd to be part of thc initiation of the carcinogenic process. Thcse authors quitc convincingly demonstrate that niathemntical modcls used for low-dose extrapolation may overestimate the risk by sevcral orders of magnitude in the presence of nonlinear pharmacokinetics. Because benrcne is converted to active metabolitics by oxidative enzymcs, thc dctermination of the levels of benzcnc that saturate U N A rcpair. detoxificationof active metabolites. and measuremcnt of DNA adducts in DNA of appropriatc tissues are really needcd before one can adopt the appropriate mathematical modcl for rclationship bctwecn exposure to bcnzcne and incidence of leukemia or ony canccr. Irvinc" clairly pointcd out lhc nccd Ibr understanding the mechnnism of bcnzcnc carcinogcncsis :ind low-dose di\ta p i n t s or thc dcbdtc will not be resolvcd. The asscrtion of Infantc et ;lI" 'I and Whitc ct al" that exposure IO benzene at I ppni is leukcmogcnic should be questioned for thc reasons discussed abovc. Lcukcmia is not the only hwiopoictic disease obscrved i n bcn?cnc-cxpossd workers. Tsonysb' hiis rcvicwed occup;ition;il l'actorsin the. cpidcmiology ol'clieinic~illyiriducrd lymphoid ;ind hclncc poictic c;i~iccrs.In her discussion of bcnrcne. S I X pointcd out thzt inultiplc nycloil~il.rcticuium I .. 4 EUGENE P. CRONKITE cell sarcoma. lymphosarcoma, and othcr Iym- is probable that these mctabolitcs also can cova- phoid ncoplasnis havc bccn obscrved in highcr lently bind 10 chromosomal DNA. Kalf ct al" than c%prctedrates. Onc can concludc from thc extcndcd thc cariicr studics of Rushmorc et ai" accumulated human expericnce that workcrs and identified several deoxyguanosinc-adducts exposed to bcnzene have a higher incidencc of suggesting that P-benzoquinone, hydroquinone. hematopoietic neoplasms. In the opinion of thc phenol. and 1,2,4benzcnetriol form adducts with author. onc cannot construct a dose-erect curve guaninc. from human exposures. Furthermore, one cannot Heidelberg~r~a'nd Millcr and ~liller'B*'9dis- exclude the possibility of D threshold of benzenc cuss in detail thc enzymatic activation of inactive exposure that is not carcinogenic. For the reasons procarcinogens to chcmically reactive electro- discusscd earlier. it is highly unlikely that thc philic species. This activation is generally csrricd "onc hit" modcl is appropriate for estimating the out by the nonspccific drug-inctabolizing micro- risk of cxposurc to bcnzcnc. somal cnzyncs using cytochrome P-450.Epos- BENZENE METABOLISM ides are the activ:ited metabolites of scvrral polycyclic hydrocarbons. Busch ct ai9 havc cxtcnsivcly rcvicwcd t hi: La\\ lcf' has studied alkyl;ition oi D Y A by mctabolisni of bcnzcnc. Bcnzcne. except at very sevcrzll known carcinoscns. Alkylation 31 0 io 6 high concentrations. has no known biological guanine is correlated with thc induction o i thy- I elTccts. At concentrations of many t houstinci moiiwt:i in c57uI/6 niicc. Alkylation 01' DS.-\ is ppm. it causes cardiotoxic and ncurologic clrccts. most likely randoni and the probability. thcrc- With the present exposure limits, thcsc are of no fore. for mutation would depend on the extent of consequence. alkylation at various sites. Lawleg believes that I About 40% of benzenc is exhalcd unchanged carcinogenesis involves specific modilkation of in the expired air. Thus 60% is converted by a DNA of target organs and thc changes asso- series of enzymes. Thc first stcp is convcrsion to ciatcd with tumorigencsis are thosc rissocirited bentcnc cpoxidc by arylhydrocarbon hydroxy- with mutagenesis through miscoding rather than lase ( A H H ) . Spontancously. about 3,370 to 507.is deletion of gcnctic material. Although onc can ' convcrtcd to phcnol. This in turn is convcrtcd to corrciatc incidcricc of tuniors with d c p c c of phcnyl glucuronide and cxcretcd in thc urine adduct formation. othcr factors not clearly along with othcr Icsscr conjugates. A small rritc- dcfincd arc requircd for a tumor to be tion of phcnol is convcrtcd to hydroquinol. exprcssed. 1 Anothcr small fraction is convcrtcd to bcnzcne glycol by epoxidc hydrase that in turn is con- I t would appcar that an intcnsivc study for presence of DNA adducts in hcniopoictic tissues ! vcrtcd to catcchol by dchydrogcnasc. Thc major of mice is in order. 1 candidates for thc activc mctabolitcs arc benzcnc ! epoxidc. hydroquinol. and catcchol. Bcctlusc EFFECTS OF BENZENE UPON HEMOPOlESiS ! major clrccts of bcnzcne arc upon the hcmato- Early studies conccntratcd on the ciTcct of i poictic progcnitors. thc qucstioii hiis bccn mkcd bcnzcnc on the periphcral blood and histology of i whether bone marrow cclls havc the cnzynics for hcmopoictic tissues. For cxtiniplc. in 1916 Scli- I converting bcnzcnc to thc activc nictabolitcs. ingMand Wcisskottcn ct ;il" obscrvcd IynipllLjpr- prob:ibly catcchol ilnd hydroxyquinone. This hiis nia. marrow Iiypcrplasirl. and scvcrc rlncmia bcen ;inswcrcd in the ntfirniativc by Andrcws ct after feeding or injecting bcntcne. There is ;In ai5 ilnd Irons ct Microsomal cytochrome extcnsivc 1itcr:iturc on the cll;zcts of' bcnLcnc on P-450 produccd phcnol. catcchol. hydroquinone. thc pcriphcral b i d Of scvcral spccics al'tcr dif- and P-bcnzoquinonc. Rushmorc et ill'' have ferent routcs of administration and amounts of rcportcd that rabbit bonc marrow mitochondria. bcnzcnc. Thcsc studics are wcll sumniarizcd by strippcd of thcir outcr mcmbranc. hiivc bccn Lcong" and Goldsrcin." Lyniphopcnia and its shown to carry out thc NADPII-dependent duration is ;I direct function. of the aniount of bioaciivation of radiolabclcd benicnc in vitro to bcnicnc administcrcd. as is ancniiri ;\nd throni- mciabolitcs that cowlcntly bind ti) mitochun- bopcnia. Ciranulocyws arc rc1;itivcly rcsiwni IO d r h i DSA. 1i1u.s inhibiting tr;inscription. Tliw i t bcn7cnc. h i v i n g ;I diininutiun onl? :iI'tcr long BENZENE LEUKEMOGENESIS 5 cxposurcs to largc aniounts of bcnzcnc. The degree of hypoplasia and its duration arc also dose dependent. The prcccding arc cKccts occurring Iatc in the hemopoietic pathway and may not be obscrved when subtle damage is induced iR stcm cells. Bone marrow cell cycle kinetics of short-term benzene exposure have been studied by Irons et al" after injection of benzene. Flow cytometry showed a relative incrcasc in cclls in G?or M phase of tell cyclc. In addition. thcrc \*::is an early incrcase in uptake of 'HTdR into DNA. This \vas followed by a decrease sugzcsting a defcct in maturation of prccursor cclls. Snydcr ct all*gave lifctimc exposure of 100 and 300 ppm bcnzcne in air six hours a day, five days a week to AKR and C57B1/6 mice. A K R mice developed anemia and lymphocytopenia. Marrow hypoplasia occurred in 20% of the benzcnc-cxposcd compared to 2%in thc control mice. Thirty-three percent ol'C57RI/ 6 iiiicc dcvclopcd granulocytic marrow hypcrpiasia coniparcd to none in thc controls. The incidence of neoplasia will be covered later in this review. Snyder et ai6' also exposed Tits to 100 ppm benzene in air for a lifctimc. Anemia and lymphopenia were significant compared to the control sham-exposcd rats. In addition, benzene-exposed rats showed marked accumulation of splcnic hciiiosidcrin compared to sham-exposed rats. suggesting heniol)sis. The prcccding studies clcirly indicate that benzene mctabolitcs arc damaging to bonc iiiarrow but do not provide insight into cellular sites of action in bonc marrow. Phcnotypic chiinpcs in hcniatopoictic cclls. such as biochemical and morphologic abnormalitics. irrcspcctivc of cause. result from genotypic alterations in thc pluripotent stcm cell that perpctuatcs hcmatopoicsis or in early progcnitors that havc little or no self-rcncwal capability. In the lattcr casc. thc phenotypic changes will bc flceting and disappear as the precursor cell and its progcny dividc. niaturc. and arc removed. In studies on hematotoxicity of pollutants. one conccntrlitcs on the pluripotcnt hcniopoictic stcni cell. its rate of proliferation and di1ycrcnti;ition into identiliable cell lines. and the mitotic potcn- ti31 of the inimcdi;ltc dcsccndants of tlic pluripotent stem cell. It is \vel1 L n w n t h J t thc pluripoicnt stern ccll can only be nssnycd in the niousc by counting the nunibcr of gross spleen colonies formcd in the fatally irradiated mousc following i-njcction of a known nunibcr of bonc marrow cclls. This technique \viis dcvclopcd by Till and McCulloch.M Thc cell colony-forming unit in the spleen is callsd thc CFU-S.Thc colonies are cloncs arising from a single ccll. Aftcr several sclf-renewing mitoses. t he colonies conimcncc to dircrentiatc among the rcd. whitc. megakaryocytic. or mixed cell lines. Aftcr seven days. thc colonics arc ciisily sccn by thc eye on the surfacc of the splccn. It has bcen shown that although thc total number of colonics sccn in thc spleen rem;iins constant. colonics arc disappearing apparently by dilfcrentiation after mctastasis into the blood at thesame rate as colonies are rcappcaring. Thesc observations. made originally by Magli et al" and later confirmsd by Priestly and Wolf." in an clcgant tcchniquc of transparcnt chambcrs in which thc splccn is visua1im.I. introduced ncw nolions into the interpretation of splccn colonies. The hematologic phenotypic altcrations in- duccd by bcnrcnc have bcen reviewed by Laskin and Goidstein4' and Snydcr and K0csis.b' They indicate injury to thc sclf-renewing CFU-S that is transmitted to thc identifiable progcny. The injury may be manifwed by a pcrmanent ch:inp in gcnotyc. in which casc thc phenotypic expression will be pcrni:lncnt. Disappcarancc of the phcnotypic expression is indicativc of cithcr rcp:iir of the genotypic injury by proccsscs yct to be identilied or dying out of the :iITcctcd clone of stct11 cclls. Uycki ct a1'" cxposcd niicc eight hours day 10 4 6 Y O ppni benzene for onc-. thrcc-, a11d cigllt- hour sessions and then incasurcd thc cellularity. CFU-S. iind colony-forming units in culture (G M-Cl..U-C) of bonc ni:irro\v. Gkl-CI-U-Cs ilrc the progcnitor cclls for franulocvtcs ;lnd macrophages. Thcy arc dctcct:ibfc by tticir :ibility to form macroscopic colonics ol' gr;inuiocy:s and/or macrophages in agar culture.'? One eight-hour exposure rcducrd the G J1-CFL-c content of marrow to 37'%0f tlir control v:1luc 111e day after the cxposurc without any ch;insc in ccllularit!. The GM-C'FU-C proprcshivcly 111crcmcd on d:iy 3. 4. ;ind 7. but \ w s still 23'.; below control>. Tlircc cight-hour exposures rcsultcd in rurthcr dccrc:iscs in G M - C W - C contcnt of tlic iiiarrow. Thrcr ci?ht-hour c s p W."'"'- .- .w. .t ._ 6 EUGENE P. CROiIKITE siircs dropped the CFC-S content of ni:irrow by nc:irIy IIhctor of thrcc. iiicasurcd 24 hours after the I p t session. The concentration of bcnzcnc. was much grc;itcr than in other studics and obscrvations were mridc fur only a few days. Gill ct ai" cxposcd mice to 4000 ppm benzcnc six hours Iday. tivc days a wcck for 6 wecks. reducing thc ratio of CFU-S to marrow cclls bv a factor of four. Thc dcvelopment of cight-day splenic colonies after rescue marrow transfusions of the fatally irradiated niousc \vas inhibitcd by continuous exposure to 100 ppm benzene in air, clcarly demonstrating that bcnzcnc mctabolitcs inhibit stcm ccll self rencwal. Grccn et ELI" studicd the etTcct of graded cxposurc to bcnzcnc in air upon stem cclls by the spleen colony technique iind upon early granulocytic-macrophage prccursors by in vitro culture. Thcir exposure rcpinirn used conccntrations of I.I:9.9:103: 300;603; 1.276; 1.416: or 4.862 pprn sis hours pcr day for five days. In another study. micc wcrc given 9.6 ppm for 50 days. In a third study. micc wcrc cxposcd to 302 ppm for 26 wccks. Fivc days" cxposurc to 103 ppm or more induccd dccrc:iscs in inarrow and splcnic ccllul&rity. in addition to dccrcascs in thc marrow and splenic CFU-S and GM-CFU-C. Whcrcas cxposurc to 103 ppm for livc days reduced CFU-S and (;M-C*I.*U-C.c x p w r c to 9.0 ppii for 50 d;iy (ncarly the rime integral dosc) h:id no effect. Thc prcvious siudics on stcm cclls and Iicniopoictic prccursors wcrc of rclativcly short durntion. Thc abiiily to rccovcr and thc fr;iction in DNA synthesis were not cxiimincd. Cronkitc ct iil''.'5*'6performed studies with two-. four-. ciglit-. and 16-wcck cxposurcs and post cxposurc up to 15 weeks. Bcnzcnc concentrations wcrc IO. 25. 100. 300. and 400 ppm for 6 hours pcr d;iy. fivc days pcr wcck. Exposure to 100 ppm or hishcr for 2 w o k s (ten exposures) reduced marrow ccllul;iritg and thc number of CFU-S in thc banc iiiarrow. Thc fr'raction of CFU-S in DNA synthcsis was signilicantly incrc:iscd; thus a smaller nunibcr of CFU-Ss w r c producing nc:irly :i normal nunibcr of blood cclls. The reduction i n CFU-S w s observed ;is c:irIy :is i\vo d;iyc ; i h cominciicing csposurc to 400 pplii ;ind w;is maxim;il at live days. at which time a signilic;iiit incrc;isc i n tlic fr;iction of C W - S in DSt\ synthcsis w;is sccn. I\ Iyrnphopcni:! was obscrwd niter ten exposures to 25 ppm bcnicnc. ;I conccntr:ition only 2.5 timcs thc pcrniissiblr: cxpusurc Icyel. \Yhcn inice wcrc cxposcd to 300 ppm for 1 or 4 weeks. thc CFU-S had rcturncd to normal levels by 1 weeks after cxposure. When exposed for 8 weeks. thcrc was a slow linear increasc in the number of CFU-S in thc marrmv reaching the lcvcl of thc sham-cxposed 16 \reeks after tcrniination of cxposure. Whcn exposed for 16 weeks. it took 25 weeks for thc CFU-S to aitain the level of the sham-exposed micc. The pcripheral lymphocyte count rcturnrd to thc level of sham-exposcd by 8 wecks after termina- tion of cxposure. irrespective of the duraiicln of exposure. Clearly. stem cells arc seriously perturbed for a Ions intcnd. Research is needed on the genc rearrangement and protooncogcne activation. Does exposure to benzene producc the same genetic rcarrangcnicnt and activation in 311 micc? I f so. why don't dl mice become leukcmic'! This will be considcrcd Iatcr under Icukcmogcncsis. Recent studies by Baarson ct a16 show a very significant decrease in the number of late erythrocytic prccursors (CFU-Es) that can be cul- tured in mice after cxposurc of C57B l /6 to IO ppm bcnzenc in air six hours a day. fivc days n week for 178 days. The CFU-Es were 5% of thc CFG-E in thc sham-cxpossd micc. The burstfuming units ( RI-U-Es) fell l o 55'2. of the IcvcI in the sham-exposed mice by 66 days. but wcrc normal at 178 days. Lymphocytcs were about 70% of normal at 178 days. Thc ancrnin was w r y modcst. only about 8.2 x IOb DL coniparcd to 9.3 x IO6 pL or 89% of thc sham cxposcd. I f rhc CFU-E is an obligatory pathway to the circui:iting rcd ccll and amplification is unch;ingcd. one would expect a severe ancnii:i of Icss tlinn I .O IO6 rcd blood CCIIS (RBCs)/pi.. One niust starch for an cxplan:ition for this discrepancy. The ret,. CFU-Es may haw il grc;itcr ninpiification through ndditiond initoscs. I f so in vivo. this niight have bccn cxprcsscd in vitro by very 1;irgc colonics t hat wcrc not dcscribcd. Irrcspccriyc the explanation of thc discrcpiincy bctwcrll dccrciiscd CFU-E and pcripher;il red count. one c:inncii ignore the fact 1h;it exposure to 10 llplii. lhc prcscrit TU',\, hiic ;i si9nilic;ini cili.ct upon hcmopoicsis. 8niirson ct al (unpub\i>hcd rcscrirch) h:iw s h o w t h 3 t consumption u t 57. ethanol in drinking w t c r cnhirnccs rlrc clTsct of I O ppni in inspired iiir. BENZENE LEUKEMOGENESIS - 7 Oilier :ippro;ichcs to the study of Iiciiiatotosicity in iiiicc h:iw been rcportcd. H;iripy:i et :il" havc comparcd long-term culturc of bone marrow friini iiiicc csposcd to bcnzcnc compared to culturcs of bonc niarrow from sham-expose3 mice. C57131/ O Tcm;iIc riiicc wcrc exposed to 400 ppm bcnzcnc in air for six hours ;I day. live d;iys a wcck for ninc or I I days. W e obscnpcd that cultures derived from bone marrow of bcnzcncesposcd micc showed a progressive reduction in the nuiiibcr of CFU-S compared to culturcs dcrivcd froni bone marrow of sham-exposed niicc. Dill'crcnt combinations of adhcrcnt cclls and rcinosulatcd cclls showcd that culiurcs containing marrow cells from bcnzcnc-cxposcd mice had a lower capacity to maintain stcm cell proliferation than normal combinations. irrcspcctive of whether benzene-exposed iii:irrow was in ihc adherent or rcinoculatcd cclls. Thcse data show that benzene inhalation produces stcni cell injury leading t i l diminished sclf rcplication. and injury to cells producing adherent population. leading to poor support of nornial swm cell self rcnewal. Carnctt et ai" studicd the effcct of bcnzenc cspowrc upon long-tcrni bonc marrow cultures with i d without addition oi hydrocortisone. Adhercnt cell layers from mice exposed to 100or -100 ppni for ninc days were dcvoid of fat cclls r c p d l c s s of whcthcr hydrocortisonc Wiis i n thc culturc. Tlicsc d;it;i indkitc that bcnzcnc inllucticcs the bcliavior ol' marrow stroinal cclls in long-term bonc mtirrow culturc. Bcnzcnc also rill'ccts B and T IyniphocFtc populations. Rozcn et ;iI5' csposcd niicc for six hours a day for six days to 300. 100. 30. and IO ppni bcnzcnc in air. Exposurc to IO ppni rcduccd the capacity of B cclls to produce colonies when stimulated by lipopolysaccharidc. Phytohcniagglutinin (PHh)-induccd blutogcnesis \viis dcprcsscd by 3 I ppm benzcnc in air. Thus. another study has s h o w an cffcct when inicc wcrc exposed to IO ppm bcntcnc in air, the currcnt pcrmissible TlYA. Pfcifer iind Irons"" studicd the cll'cct of bcnzcnc mctabolitcs upon P H A stimulation of rat bonc marrow. Hydroquinone inhibitcd mitogenesis. The prcscncc of niicroniolar amounts of mctabolitc (p-benzoquinone :ind I .2.&bcnzcnctriol) partiall! inhibited PI I . \ stimulation. I n earlier studies. Grccnlcc ct a ~ " and Rickcrt et ;iI" showed tli;it bcn7cnc taxicity of hciiiopoictic tissucs is ;issoci;itcd w i t h the conccnt r:i t ion 0:'h ydroqu i none and c:i tcc hol. but not pllcnol. \!`licrc::s those studies suyycst that thc ilhovc bcnzcnc metabolites may be thc x t i v c ;igcnts. thc studics do not climinatc bcnzcnc cpoxidc. a short-lived precursor of the ;ibovc nict;lbolitcs. as beins ;in import:int rc;ictivc metabolite. ;$`'Cytogcmtic cll`ccts of bcnzcnc hnvc been stud- ied. Tice ct e x p c d mice to 3.100 ppm bcnzcnc in air for four hours and induced a significant incrcasc in sistcr chromatid exch;ingcs in bonc m;irrow and inhibited cell prolifcration. Prctrcatrncnt with phcnohirbitol. an inducer of P-450 cytochrornc osidases, increased thc cKcct on sistcr chromatid cxch:inges iind ccll proliferation and induced chromatid-iypc chromosomal abcrratioti. Gad-El-Xnrini ct ai" found that benzcnc induces ;in incrcasc in micronuclei of polychromatic normoblasts. The cffcct \\`iiS incrcascd by induccrs of P-448monoxydasc but not by plicnthdrbitol. ;in inducer of P-450. LEUKEMOGENESIS Cronkitcl` in I96 1 bcforc discovery of protooncogcncs bclicvcd that any tigent that produced marrow apliISi3 \+pould be leukemogenic. Witti morc sophisticntcd knowledge on carcinogcncsis. thcrc is a $rowing body of cvidcncc that indicates lcukcrnogcncsis is I niultistep. compicx process. Tlicrc ;ire scvcr;iI types of 1cukcnii;i that i n turn suggest that tlicrc :ire dillkrcnt iiioIccuI:ir iiicch;inisiiis involved or t h a t the saiiic niolccular mech;inisiiis ;ire induced in dill;.rcnt cell lincs or at dill'crcnt stages of dill'crcntiation in ;I given cell line. There is cstcnsivc evidence Vor the role of key gcncs i n carcinogenesis by Bidhop.' tieldin and Wcstcrmark." Cooper and Lane." Klein and Klein." and Imnd ct al." The notion of carcinogenesis being a multistep proccss is not ncw. Burcli8 dcduccd fhis on the blisis of ;I iii:ithcni:iticnl :in;ilysis of radiation-induced cancer. Wliercas thcrc is ovcrwhclming cvidcncc iniplicat ing thc ;ictivation of protooncogc1ics to act ivc oncogenes with production of I hc one,)genic proteins t h t i n wine inst;inc:csw e h u l n r l l t l pous ivitli growth rilctors. tlic iiiultipiicity ,)I'itlc. gcncs. tlicir products ( protein kiiiahch), rcceptors. ;inif i~itcrnctiuiis.prcscni :I wry coiiipIic;ltcd picture tIi;it is not ;15 yet unr:iwlcd. Lcukcnrogciicsis is clcarl! ;i multistep process involving. -_.I__ . . -, , . .. . ... .- I I 8 EUGENE P. Ci(0NKITE amcng othcr fiictors. ;i random production of D N A adducts. Thc hctcropity of abnormalitks is well-rcviewcd by Francis."' She prcsents a hypothetical mcchanism for induction of myloid leukemia in man involving activation of the tyrosine kinase group of oncogenes. impaired diner- entiation. and imparts a role to D N A topoisom- erasc. the DYA untwisting enzyme. Inhibition of DNA topoisomerase freezes cclls in the ccll cycle. but docs not kill them and apptxrs to be involvcd in indmion of sister chroni:itid exchangcs. This enzymc is involved in D N A rcpair and dcfcctivcfunction may rcsult in dcfcc- tivc repair. Whcrcas onc cannot yet piccc together all of the scqucntid stcps rcquircd to initiatc and produce a Icukcmia. it is c h r th;it animal models for induction of leukemia to be described next arc available for intensive study of molecular changcs in DNA during Icukcmopcnesis. Farbcr" dcscribcs thc ccllular scqucncc of cvcnts in c;ircinogcncsis t1i;it iii:ry be helpful in designing cxpcrirwnts on the murinc models to be dcscribed. Exposurc of mice to inhaled bcnzcne has contributcd substantially to our practical knowlcdge on exposure conditions that arc Icukcrnogcnic in dill'crcnt str;lins of micc. Stoner ct alM cxposcd hairless micc to bcnzcne throughout thcir whole lifc. The incidcncc of Icukcmia was not incrcascd. In Fact. ;in in1crprct;ltion of ihcir data suggests that exposure 10 bcnzcnc by inha1;ition may havc supprcsscd the dcvclopnicnt of Icukcmiri. Snydcr ct :iI6' cxposcd CS7B I / 6 and A K R niicc to 300 ppni 67 hours n day. live days a wcck for a lifctirnc. Thcrc was a significant incrcasc in thc incidcncc of Icukcmi;i/lymphoma. In studies on rats cxposcd to 100 ppni for ;I lifctimc. a sing!c casc of chronic myclogncous Icukcmia, a rarity in thc rat. was obscrvcd by Goldstein et Furthcr siudics by Snydcr CI ai6' shotvcd scvcrc hcniatotoxicity of inh:iling 100 ppni bcnzcnc in air. but no siknificant ilicrcasc in hcmopoictic ncoplasms. Again, rare ncopiasmsof the rat wcrc sccn. suggesting a ncopl:istic etTcct. Maltoni ct summarizing rn:iny ycars of investigation. h:ivc produced strong cvidcncc that bcnzcnc by injcctiorr and inscstion produccs an cxccss nuiiibcr of dillcrcnt tumors in thc rat. Thcy cmphasin thc nccd for dosc-clrect studics to cstablish thc rciationship bcttsccn csposurc and neoplastic incidcncc. Cronkitc et al"'-'5.!5npproitchcd t h t problcni of bcnzcne leukcrnogcncsis using CSiBI !;6 male mice kno\vn 11) harbor the radiation icukcnia virus that is activated by exposure to iuniting radiation and the CBI\/C;l maie mousc. which has a very low spontancous incidcncc of 3CUtC myeloblastic leukemia ( A M1,) and has been shown by Major and Mole'* to d c v e l q ~3 high incidencc of AML after csposiire to ionizing radiation. Thc exposure regimen was bascd on the apparent exposure conditions of human beings described by Aksoy et allA and Vigiiani and Saita." Human bcinp do not commence work in industry until 18 years or oldcr. Thus. exposure of micc \vas comnicnccd at 12 io 14 w c k s of a p . whcn they are sexu;illy mature. I t was assumcd thal human beinzs wcre cxposcd for about I S 5 of thcir livcs and. accordingl!. micc w r c exposcd for 16 \vceks. approximately 15% of thcir lifcspan. Thc work wcck was mirnickcd by cxposing inkc sis hours :i day. tivc d:iys a weck. \ - h e n C57Bll6 female inice wcrc cxposed to 300 ppm bcnzene and hold for lifeiinic study. a highly significant earlier onset of thymic lymphomata. nonthymic lymphoma. and Icukcmia WIS observed. Male C57BI/b hnd ;I morc rapid onset of hcmopoietic ncoplasms. In both sexcs. the ultimatc 1ifc:imc incidence was nearly thc some (unpublishcd data). When CB:\/Ca malc micc wcrc cxposcd lo 300 ppm for Ih wccks. a strikins diffcrcncc bctwcrn the bcnzenc-cxposcd and the sham-cxposed micc \v;is sccn. A M L comrnenccd appearnncc nt about 2 I O days of age or 2 weeks after termination of exposure. A t 600 days of agc. the incidcnce of AML was 38% and incrcnsing with no n h l L in the sham-cxposcd micc. \Vhen fcnialc CBA :Ca micc wcre cxposed to 300 ppm or IO0 ppm for 16 wccks. a highly significant incrcasc in ncoplasnis was obscwcd. pmiciil:irly mammary adcnocar- cinoma. Thcsc studics arc not 3s yct coniplclc. The sh:ipc of the iiicidcncr curvcs wggcns that thc ultiniatc incidcncc cf acute Icukcmia may approact1 50% coniparcd to less th;m 5 5 in the sham-cxpascd niicc. Thc timc scqucncc for appcarancc of ;~ciitcIcukcmia is cstriblishcd. It appclirs \bat thc CDr\/Ca m : h mouhc is an appropriatc animal in which onc colild dctcrmine the abundance of DNA adducts in hcmopoictic tissucs with timc nnd idcntify the ;ictiwion uf protwncor,cncs to provide beiicr insight into I h r BENZENE LEUKEMOGENESIS. - 9 mcchanisni of bcnxnc Icukcmogcncsis. In par:illei. otic could cfctcnninc if the iticidcncc versus bcnzcnc cxposurc has a sirpilar dosc-cil'cct rclationship to DNA adducts. I f both h;ivc thc s;iIIic mathematical relationship. one would have ;I rcsonablc cxperirncntnl basis for intcrprctation of low-dose cxposurc of man. CONCLUSIONS Our conclusions arc as follows: ( 1 ) Bcnzcne is lcukcmogcnic in inan and mice. (2) tlic dosc- clTcct rclationships have not bcen cstablishcd in man or mousc. (3) thcrc arc adcquntc rcnsons to qucstioti t tic "oiic hit'. tliodcl lor prediction of low-dosc cil'ccts of inhaliltion of bcnzcnc. (4) bcnzcnc mctabolitcs producc many toxic clfccts upon hcmopoicsis that arc dose dcpendcnt. ( 5 ) bcnzcne inhalation of IO ppm has bcen shown to producc ctrccts on early crythrocytic prccursors. The magnitude of thc cffcct is not compatible with the slight iincmi;i. and ( 6 ) tllc prcscncs of hcrnopoiclic clfccts after cxposurc to IO ppiir bcnzcnc in air rcquircs furthcr investigation and considcr:ition of whether thc present T W A of IO ppm is justified in the workplacc. REFERENCES I . ;\kso! M. 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Major IR. ,\lolc KI1: M i e l t i i d Irukemia in CB.4 mice. h';rturc 272:J5!3-456. 197X 51. 0 1 1 !W.Twn\cnd JC. I:i,hbcck \VA. ut 1 1 : Vtiriahiy ;inion&! indi\du& c % p ~ ~irirblcn/cnc. Arch 1:nvircin tlc-lth 33.3-lO. 197s . 52. pfciicr R\\`. Irons RD: Inhibition of'lcctin-silmul;,irJ Iylnphoc+ :iggluiinaticrn and iiritogcnesis by h!droquin\mc. Rcactivit? witti intracellular sulfhydryl compiunds. Tuhicologisi I :106. I98 I 53. Pfeifcr K\Y. Irons RD: EITect of bcnzenc mctabulittb on PlIA h n u l a t c d Iymphopiiesis i n rat bone marrow. J Reiiculcxndothcl SOC31:155-170. 1982 533. Priestley GV. IVolCNS:A technique for daily rwni- nation of splccn colonics i n micc. Exp tlcmatol 1 3 : 7 3 ! - i 3 . IYXJ 54. Rinsky RA. Young RJ. Smith AB: Lcukrmia in benzcnc workers. Am J Ind Med 2217-241;. 19sI 55. Richcn DE. Baker TS. Bus JS. CI al: Bcnzcnc disposi- tion in the rat after exposure by inhalation. Toxicol :\pi4 Pharmacol J9:4 17. I98 I 56. Rinsky RI\. Young RJ. 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Toxic01Appl Pharmicol J4323-331. l9BO 63. Snlder CX. Goldstein BO. Scllakumar t\R: Evidcnce four hematotoxicity and tumoripncsis in rats exposed 10 1009 ppm bcnicne. A m J Ind 4Med 5:429-134. I ? ~ J 64. Stoner RD. Drcw RT. Bernstcin IN:Bcnzcnc inhJ1;ition ctfects upDn tetanus anritoxic respnses and lcukcnicr. genesis in itiicc in I'rlxccdin~s of ihc 20th Ildnl'cird 1.ite Sciences S v n i p i u m an Cod! Conversion gnd the Environ. mcni. Oak Ridge. TN. Technical Intornintion Ccnisr. I ~ S O . pp 45 1461 ,,[65. Ticc RR. COS^:^ DL. Drew R T C!titScnclic elreels inhaled bsn7cne i n murille bune marrl>\v. Induction ,)I sister chromatid exchanges. chromosomalaberrations. Jnd cellular prolifcraiion inhibitiun in I)B;\/2 micc. Prac hati .\cad sc, USA 77:214X-?l52. l9NO 66. Till .IF. ~ I ~ C u l l Et A~ :~Ah dircrt msasurrnrcnt ,,I r:idi;itiun hcnsitiriiy ti[ norin:il mouse buclc marrou ce~l,, Radiat Kcs I4:ll3-227. 1961 67. Tsongm TA: Occup:iiion;~l factors in the vplricliiii,,p! of chcminlly rnduccd lymphold and heiiiupietic cancer, in Irons R (cd): Toxicology of the Blood 2nd Ronc \larrrlu. Ncu Ywk. kivcn. 19S5 68. U!ckr EM. h l l k a r AI:. Shwm;in I N ' , CI 21: .\cutg toticit> of bcn/cno inh;iIaiian i n hcmnpxitc rrcuurrttr ~ C I I \ Tii~tcul,\ppI l'li.irnrxbd WJ'3.57. IY77 $ BENZENE LEUKEMOGENESIS c - 11