Document mqMwxyB32kMk6pjvZmOKLxevQ

Environmenral Health Perspectives %I. 100,pp. 293-306,1993 The Toxicology of Benzene by Robert Snyder,'Gisela Witz,' and Bernard D. Goldstein' Benzene is metabolized, primarily in the liver, to a seriesof phenolic and ring-openedproducts and their conjugates. Themechanismof benzene-induced aplastic anemia appearsto involve the concerted action of several metabolitesacting together on early stem and progenitor cells, aswell ason early blast ceUs,suchas pronormoblastsand normoblaststo inhibit maturation and amplifmtion. Benzene metabolitesalso inhibit the functionof microenvironmentalstromalcells necessary to support the growthof difkrpntiating andmaturing marrow cells. Themechanismof benzene-inducedleukemogenesis is less well understood.Benzene and its metabolitesdo not function wellasmutagensbut BIP highly clastogenic, producing chromosome aberrations, sisterchromatid exchange, and micronuclei.Benzene has been shown to be a multi-organ carcinogenin animals. Epidemiologicalstudies demonstratethat benzeneis a human leukemogen. Them is need to bet- ter define the lower end of the dose-responw curve for benzene as a human leukemogen. The application of emerging methodsin biologically based riskassessmentemploying pharmacokinetic and mechanisticdata may help to clarify the uncertaintiesin low-dose risk assessment. Introduction The biological impacts of benzene have been studied in humans and in animal models for most of this century.This review concentrates on several aspects of the biology of benzene. We discuss its metabolic fate, our current understanding of the mechanism by which it produces its effects, attemptsto develop pharmacokinetic models for its disposition, and, finally, we discussthe impactof benzene on humans. Our aim is not only to present a synopsisof the literatureon benzene toxicity, but also to challenge investigatorsto initiate new studies that will lead to a thorough understandingof benzene as a hazardous agent in our environment. Metabolism Benzene is the smallestand most stablearomatichydrocarbon. In order to be toxic, benzene must be metabolized to reactive intermediates (1-3).Available studiessuggest that benzene toxicity is mediated by multiple metabolites acting on multiple cellular targets ( 4 ) .This discussionbriefly reviews benzene metabolism, mechanism of metabolite formation and the chemical reactivity of benzene metabolites in relation to cellular targets. For recent detailed reviews, the reader is referred to Kalf ( 5 ) ,Snyder and Chatterjee ( 6 ) ,and Yardley-Joneset ai. (7). 'Environnientaland Occupational HealthSciences Institute,Rutgers,the State University of New Jersey and the University of Medicine and Dentistry ofNew JerseylRohert %od Johnson Medical School, Piscataway, NJ 08855-1179. Address reprint requeststo R. Snyder,E n v i m n t a l and Occupational Health Sciences Institute. Rutgers,the StateUniversity of New Jersey and the Univer- sity of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School. Piwataway. NJ 08855-1179. Metabolic Pathways The major metabolism of benzene in vivo takes place in the liver and consists of the formation of ring-hydroxylated compounds (Fig. 1). Early studiesconducted in rabbits(8-10) showed that benzene is hydroxylated to phenol, catechol, hydroquinone, and 1,2,4-benzenetriol,which are excreted as ethereal sulfatesand glucuronidesin the urine (Fig. 2). Work by Jaffe (11) and other investigators (12)showedthat benzene metabolism also involves ring opening as indicated by the urinary excretion of tmns,tms-muconic acid, a ring-opened, six carbon dienedicarboxylic acid. The formation of tmns,tmns-muconic acid (muconic acid) was definitively established by Parke and Williams (9),who demonstrated theexcretion of [14C]muconicacid in the urine of rabbits dosed with [I4C]benzene. The metabolicpathways of benzene originally determined in rabbits were subsequently establishedin rats and mice (13-16). Two metabolic pathways appear to be involved, one leading to ring-hydroxylated compounds and one that involves ring opening of benzene. The hydroxylated compounds are further metabolized to glucuronide or sulfateconjugates, which are excreted in the urine. Theseconjugatesare detoxificationproducts, because conjugation leads to their eliminationand prevents the formation of toxic intermediates derived from hydroxylated benzene metabolites. Conjugationwith glutathione and urinary excretion of the mercapturicacid is an additionalpathway that detoxifiesbenzene oxide,the initial reactive intermediate formed during metabolism of benzene to phenol (discussedbelow).The urinary metabolite muconic acid is at present the only known ring-opened metabolite formed from benzene in vivo. This metabolite is a detoxification product of fmns,rmns-muconaldehyde, a reactive intermediate formed during microsomal metabolism of benzene via ring opening (17). The initial step in the metabolism of benzene results in the for- * 294 SNYDER ETAL. m-0 BENZENE FIGURE I. Intermediary metabolism of benzene. [O]indicates oxidation without specifying mechanism; GSH,glutathione. Question marks indicated suspected. but as yet unproven. pathways. Dotted lines leading to 1.2.4-trihydmxybenzne suggest that it may be formed by either pathway. Glucuronide and Sulfate Conjugates Mercapturic A d d s C~-CH-cOcm F-- &N-ACETYLCYSEWYL-S2.4-CYCU)HEXAMENOL C*CH--COOH &I NH--COCY PHENYLMERCAPTURIC ACID on NHCOCH, HYMIOOUlN0NE.SPHENYLMERCAPTURIC ACID DNA-based Adduct(s) N-7-PHENYLGUANINE Ring-Opened MetaMite(s) HH Hooc-c =cI -c =cI -COOH II HH TRANS.TRANS-MUCONICACID FIGURE 2. Urinary metabolitesof benzene. The sulfate and glucuronideconjugatesof phenol, catechol, and hydroquinone have been characterized. The location of the conjugateon 1.2.4-trihydroxybenzene has yet to be established.The glutathionederivatives. the DNAbasederivative and thering-openedproduct are those which have been identified in urine. mation of phenol. Numerous in vitro studies utilizing cellular fractionsor reconstituted purified enzyme systemsindicatethat this step involves metabolism of benzene by cytochrome P-450 monooxygenase (18).Benzene is a substrate for isozymeP-450 IIEl (19), and P-450 monooxygenases are also active in the metabolism of phenol to hydroquinone and catechol (20).The formation of phenol is believed to involve the intermediate for- mation of benzene oxide followed by rearrangement to phenol (21).Alternatively, phenol can alsobe fbrmed from benzene oxide by acid-catalyzed opening ofthe epoxide ring, followed by TOXICOLOGY OF BENZENE 295 aromatizationvia loss of a proton. Mechanisms of phenol formation that do not involve an in- termediate epoxide but rather direct insertion of oxygen or aromatic hydroxylation(22)may alsoaccount for the formation of appreciable amounts of phenol. Phenol formation from benzene in a reconstitutedsystem containingrabbit liver P-450 isozyme LM2 (P-450IIEI) or microsomeswas reported to be inhibited by the hydroxyl radical scavengers mannitol and dimethyl sulfoxide (DMSO), as well as by catalase, horseradish peroxidase. and superoxidedismutase (23).These findings suggest that the cytochrome P-450-dependent formationof phenol can be mediated by hydroxyl radicals most likely generated from hydrogen peroxide. The latter is thoughtto be formedby dismutation of superoxide anion radicals released from cytochrome P-450. Hydroxyl radical-mediated phenol formation involvesaddition of a hydroxyl radical to the benzene ring, resulting in the formation of a reactive hydroxy cyclohexadienyl radical (24). Phenol can subsequentlybe formed by a dismutationreaction as suggestedby Cohen and Ofodile (25),who investigated phenol fonnation from benzene in a hydroxyl radical-generatingFenton system. A role for hydroxyl radicals in the metabolismof benzene to phenol hasalsobeen suggested by Gorsky and Coon (26),who concluded that the hydroxyl radical-mediated formation of phenol is thedominant pathway at micromolar concentrations, whereasat higher (millimolar)concentrations,thedirect oxidation by P-450 is quantitativelyof much greater importance. CytochromeP-450mediated metabolismof phenol leadsto the formation ofhydroquinoneand catecholand the subsequentformationof 1,2,4-benzeneuiol.An alternativepathway for the formation of catecholinvolvesmetabolismof benzene oxidebyepoxide hydrolaseto benzene-rmns-dihyddiol, which is converted to catecholby the actionof a dehydrogenase. Benzeneoxidecan also react with glutathioneunder the influence of a glutathione transferasetoeventuallyyield a prephenylmercapturicacid (6-NacetyI-cysteinyl-S-2,4~lohexadienolU).nder acidicconditions this metabolite is aromatized by dehydration to S-phenylmercapturic acid, and both compounds have been observed in urine (27).The hydroxylatedaromaticbenzene metabolites are further metabolized to sulfate or glucuronic acid conjugates. The metabolite 2-(S-glutathionyl)hydroquinonehas been identified in microsomal metabolism mixtures incubated with benzene or phenol in the presenceof added glutathione (28.29). It is presumably formed by the reactionof glutathione with benzoquinone,a reactive intermediatederived from the oxidation of hydroquinone. Hydroquinone-S-phenyl mercapturic acid derived from the metabolismof2-(S-glutathionyl) hydroquinonehas been identified as a urinary metabolite in rats dosed intraperitoneally with benzene, phenol, or hydroquinone (30). Thus, Figure 2 shows that the urinary metabolites of benzene may include theglucuronicand sulfateconjugatesof the phenolic metabolites, the premercapric and the mercapturicacids derived from benzene oxide, the mercapturic acid derived from pbenzoquinone, N-7-phenylguanine,derived from a DNA adduct of benzene, and muconicacid. Microsomal metabolism of benzene leads to the formation of rmm,rmm-muconaldehyde(Fig. 3), a reactive nng-opened, sixcarbon diene dialdehyde(27).Muconaldehyde was also shown to be formed in aqueoussolutionsof benzene irradiated with X- iy ADH 4 __ c CHtOH 6-HYDROXY-2.4- TRANS-TRANS- \HEXADIENAL TRANSTRANSMUCONALDEHYDE 6-OXO- TRANS,TRANSHEXADIENOIC / ACID 1.6-DIHYDROXY-2.4TRANSTRANSHEXADIENE 6-HYDROXY-2.4TRANS.TRANS- HEXADIENOIC ACID t TRANS.TRANSMUCONIC ACID FIGURE 3. Intermediary metabolism of muconaldehyde. ALDH, aldehyde dehyclroguuw;ADH, alcohol dehydrogenase.The pathway with the question mark indicates a possible route of metabolism. rays (32,32)presumably via hydroxyl radical-mediatedopening of the benzene ring. These latter studies and others on the hydroxyl radical-mediated formation of phenol suggest that free radicals may participate in benzene metabolism. The metabolismof muconaldehyde(MUC; Fig. 3) by mouse liver cytosol or a mouse liver solublefraction results in a variety of metabolitesthat are formed by oxidation andlor reduction of the aldehydefunctional gmup(s) (33-35).Initial oxidationor reduction leads to the aldehydeacid and aldehydealcohol analog of MUC, respectively. Subsequent reduction of the aldehyde group of the mixed-aldehyde acid MUC analog or oxidation of the mixed-aldehydealcoholanalog of MUC leadsto the formation of the mixed-hydroxy acid MUC analog, a metabolite in which one aldehydic functionalgroupof MUC is reduced to the alcohol and one is oxidized to the carboxylic acid. Interestingly, the initial reduction to the aldehyde alcohol MUC analog is reversible (G.Witz, unpublisheddata). The mixed aldehydecarboxylic acid analog of MUC is further metabolized to muconic acid, a urinary metabolite of benzene (9.36,37). Reactive Metabolites in Benzene Metabolism The major metabolism of benzene takes place in the liver where it is mediated by cytochrome P-450 monooxygenase. Metabolismof benzene by cytochromeP-450 results in the formation of benzeneoxide, an electrophilic reactive intermediate. Evidence that benzene is indeed metabolized in vivo to benzene oxide comesfrom studiesby Mueller et al. (38)who identified N-7-phenylguanine in the urine of rats exposed to 500 ppm benzene for 8 hr. This adduct is most likely formed by reaction of benzene oxide with the N-7 of guanine, followedby aromatization of the benzeneringvia loss of a moleculeof water and subsequentdepurinationof the adduct fromDNA. Although benzene is also metabolized to phenol and other hydroxylated metabolites in thebonemarmw (39),P-450mediated metabolismin thistissue occurstoarelativelylimitedextent.Thisisduetolowlevelsofthis enzyme system in the bone marrow compared with liver. 2% SNYDER ETAL. Indirect evidence indicates that muconaldehyde may be another reactive metabolite formed in the liver (17).In addition, the reactive intermediatesp-benzoquinone and o-benzoquinone may be formed in this tissue by oxidation of hydroquinone and catechol, respectively.These reactive quinone metabolites and muconaldehyde are direct-acting alkylating agents capable of reacting with cellular nucleophiles, including DNA (40,41). Numerous in virro and in vivo studies indicate that these metabolites are genotoxic (42-45).Once formed in the liver, it is unlikely that they survive long enough to reach the bone marrow. It is possible, however, that reactive benzene metabolites, similar to other reactive intermediates, may be bound to carriers that release them after transport to the target tissue. Reactive intermediates may also be conjugated to form less reactive intermediatesor "storage forms" which, after transport, release the reactive metabolitethrough deconjugation.A third possibility germane perhaps to muconaldehyde may consist of metabolism to a less reactive compound, i.e., a mixed aldehyde alcohol MUC analog (Fig. 3), which itself may be hematotoxic or is subsequentlyreactivated in the target tissue by metabolism to the original reactive metabolite, Le., muconaldehyde. The bone marrow has been shown toaccumulatehydroquinone and catechol (46,47),two phenolic metabolites of benzene. As discussed above, bone marrow, unlike the liver, contains low levels of cytochrome P-450and high levels of peroxidases. Phenolic compounds are excellent electron donors for the peroxidase-mediated metabolism of hydrogen peroxide. Studies by Eastmond et al. (48,49)and Sadleret al. (50)have shown that phenol, catechol, and hydroquinoneare excellentsubstrates for myeloperoxidase, a peroxidase present in high concentrationsin the bone marrow. During myeloperoxidase metabolism, these phenolic metabolites undergo one-electron oxidation and covalent binding to protein. The reactive species involved in the binding are presumably radical intermediates, such as the semiquinone free radical of hydroquinone, which are formed during one-electron oxidation. Para-benzoquinone is a reactive a,Punsaturated diketonethat has been implicated in bone marrow toxicity (51,52).Phenol and catechol have been shown to stimulate the myeloperoxidase dependent conversion of hydroquinone to p-benzoquinone (5253).Based on these studies, Smith et al. (54)proposed a twostep mechanism of benzene toxicity that involves metabolism to phenolic compounds in the liver, transportand accumulationof these compounds in the bone marrow, followed by myeloperoxidase-mediated conversion of hydroquinone to pbenzoquinone in a process stimulated by phenol and possibly catechol. This mechanismofp-benzoquinoneformationdoes not exclude a similar involvement by prostaglandin (H)synthetase and eosinophil peroxidase in benzene toxicity (55). Mechanismsof Benzene Toxicity The most frequently observed toxic effect of benzene in humans and in animal models used for the study of benzene toxicity has been bone marrow depressionleading to aplasticanemia (14).The correlation between the effects of benzene exposurein humans and the effects produced in animal models was demonstrated by Santesson in 1897 (56),Selling in 1916 (57) and Weiskotten et al. in 1916 and 1920 (58,59). As a result many studies in animals have been aimed at uncovering themechanism of benzene toxicity for the purposeof applyingtheseobservations toward an understanding of the human disease. During the latter half of this century, it has become apparent that benzene alsocauses chromosomalabnormalitiesindicative of genetic damage (60-64)A.ttempts to demonstrate mutagenic activity of benzene have been largely unsuccessful (42),but benzene is a human leukemogen (65) and causes sdid tumorsin animals (66-68).Unfortunately,there is no convenient model for studyingthe mechanismsby which benzeneproducesneoplastic effects in animals. Although each of these effects, Le., aplastic anemia, chromosome damage, and carcinogenesis, are indicativeof the ultimate impact of benzene on bone marrow, the relationshipbetween these phenomena and the mechanismsby which they are initiated remain to be fully understood. This section describesthe effects ofbenzene in model systems, discusses the mechanisms by which these effects occur as we currently understand them, and points out data gaps, which when filled may help us to a better mechanistic appreciationof the biological impact of benzene. Hematopoiesis The mechanism of benzene toxicity has ben examined on the basis of its effects on stem and progenitor cells in the bone marrowas well as its effectson the hematopoieticmicroenvironment. Hematopoietic stem cells in the bone marrow give rise to the major cell types in the circulation, i.e., erythrocytes, thrombocytes, and the several major subdivisions which comprise the leukocytes (69).The self-renewingstem cells which reproduce themselves, also yield a cell committed to eventual maturation to a circulating cell. Growth factors direct committed cells to enter the maturational process, e.g., in the redcell line thecommitted erythroid cell is sensitive to the initiating action of erythropoietin. To assure that the level of mature cells is constant despite the regular destruction of aged erythrocytes requires that the marrow provide a mechanism for both maturation from the morphology of theprimitivestemcell to that of the maturecirculating erythrocyte, plus a mechanism for amplification to sustain the requisitenumber of circulatingerythrocytes,i.e., approximately 5 million ceUs/pL in humans with a survival time of 120days. Thus, a complex series of biosynthetic eventsresults in the passageof thesecells throughseveral defined morphologicalstages, during many of which the cells undergo mitosis to increase total numbers. A related, but unique pmess leads to megakaryocyte productionand eventual release of platelets in large numbers (approximately250,OOO/pLin humans with a survival time of 10 days). Fewer leukocytes,e.g., between 5 and l2,OOO cellslpL in most species,are required. However, the process iscomplicated by the need for several different forms includinggranulocytes, lymphocytes, monocytes,etc., each dividedinto severalfunctional andmorphologicalsubtypesand with differinglifetimes. These also undergo a series of steps directed by cytokines or other growth factors involvingboth maturation and amplification. Thehematopoietic microenvironmentis composedof a variety of cells and structures including endosteal cells, fibroblasts, reticuloendothelialcells (monocytesand macrophages),fat cells, and bone and sinusoidsof the marrow and is responsible for sustaining stem and progenitor cells. Proteins such as erythropoietin, colony-stimulatingfactors, the interleukins and related cytokine mediators are products of microenvironmental cells and TOXICOLOGYOF BENZENE 291 play key roles in directingthese events (69).In the absenceof the microenvironment, primitive blood cells cannot mature. Effects of Benzene Many studieshave shown that treatment of wholeanimals with benzene leads to decrements in the levels of circulating blood cells (14).Mechanistic studies have benefited fromthe development of a variety of in v i m techniques. Thus, the bone marrow of animals exposed to benzene using various exposureprotocols can be removed and grown in culture to explore the effects of benzene on specific cell lines at more or less specific stagesof maturation. Several laboratories have reported on decreases in the multipotential hematopoieticstem cell (spleen colony forming unit or CFU-S) in mice after a variety of exposureprotocols (70-73). Similar effects have been observed using the early granulocyte-macrophageCFU-C assay (72,74)D.ecreases have also been observed using the erythroid colony-hrming unit (CFU-E)and the burst-forming colony unit (BFU-E) assay, each of which defines a committed stem cell early in erythrocyte development (X76).Exposure of mice in utero to benzene at relatively low concentrations resulted in decrements in bone marrow colony forming units postnatally (7778).These studies indicatethat benzene exertsa significant impact on both primitive and early committed stem cells. In additionto the effectsof benzeneon stem cellsand commit- ted stem cells, both morphological and functional evidence has linked benzene to impairment of the processesof maturation and amplification. Pronormoblasts, normoblasts, and reticulocytes appear successively at 24,48, and 72 hr after reinitiation of red cell development by erythropoietin (79). Synthesis of hemoglobin, including the incorporationof iron, occursonly between the pronormoblast and reticulocytestage of cellular differentiation (80,8J).One method to observe the effects of chemicals on various stageof erythropoiesisin viw involves administrationof 59Feintravenously followed by sampling 1 day later of the red cells for incorporation of the iron into hemoglobin (82,83).This provides a functional measurement of the reticulocyte pool. Treatment with chemicals at various times before iron administrationresulted in a decreasein iron uptake. These data indicate an impairment in red cell development, provided it is known that the chemical did not interrupt reticulocyte hemoglobin synthesis. In the caseof benzene (82,83)hemoglobin synthesis proceeds normally. The maximum decrease in iron uptake followingadministrationofbenzene occurred 48 hr before iron administration, a time at which the reticulocytesareat the earlier pronormoblaststage of development. Normoblast development was also inhibited but no effect was observed in these studieson committed stem cells nor on reticulocytes. Taken together, these studies suggest effects of benzene at both the stem cell and the progenitor cell stages of erythropoiesis. Studies by Dexter et al. (84) established a method for the evaluation of the hematopoieticmicroenvironment. The system allows for the growth of an adherent cell layer of stromal cells in liquid culture. Stem cellsadded to this prepahtion begin the process of maturation and proliferation supported by the stromal cells. Using this method, Garnett et al. (85)exposed mice to benzene at various doses, removed the bone marrow, and grew adherent layers that supportedthe growth, but not the differen- tiation, of colony-formingunits from control mice. Inhibition of differentiation may have been due to the failure of these preparations to grow hydrocortisone-induced fat cells essential to stromal-supporteddifferentiation.Frashet al. (86)exposedmice to both radiation and benzene and injected normal mouse marr o w cells. Using the CFU-S technique, they reported failure to restore m a r m function(86).Incubation of bone marrow cells with benzenebefore injection into irradiatedmice had no effect on spleen colony formation. Frash et al. (86)concludedthat the hematopoietic microenvironment was damaged as a result of benzene treatment. Effects of Benzene Metabolites It appears that benzene toxicity occurs only under conditions that permit the production of benzene metabolites.Thus, animals were protected from benzene toxicity after partial hepatectomy, which reduced total benzene metabolismin vivoand resulted in a decrease in d e n t bindingof metabolitesin bone marrow (2). his was not the first suggestionthat benzene metabolites play a critical role in toxicity. F'arke and Williams( 9 ) ,in their pathfinding report suggested that the phenolic metabolites of benzene might be responsible for benzene toxicity. A number of investigators subsequently attempted to identify the toxic metabolites of benzene. Both Bolscak and Nerland (3)and Snyder and co-workers (87-89) have studied the effects of benzene metabolites on 59Feuptake in mice and found that hydroquinone and catechol(50-100 mgkg) are more potent in decreasing iron uptake than phenol. Pam-benzoquinone, the metabolic product of hydroquinone, is more potent than hydroquinone. Muconaldehyde andp-benzoquinonewere effectivein inhibiting iron uptake in the range of 1-4 mg/kg. Several studies have focused on the possibility that benzene toxicity is the result of synergistic interactions among benzene metabolites. Eastmond et al. (49)studiedthe effect of administering a combination of phenol and hydroquinone and reported that the combinationenhanced the abilityof each to inhibit bone marrow function. Possible explanations for the interaction be- tweenhydroquinone andphenol includethe preferentialconjuga- tion of phenol by sulfateand glucuronide leading to a greater ef- fectiveconcentrationof freehydroquinone, the stimulationof the production of reactivemetabolitesfrom hydroquinone by phenol, or both. The same interaction was also observed in Snyder's laboratory (87,88u)sing the s9Feuptake technique. Guy et al. (89)studied the effects of combining muconaldehyde plus either hydroquinone orp-benzoquinoneon iron uptake into erythrocytes. They reported that when the binary mixtures were administered together at doses of either which were ineffective in reducing iron uptake, the combined effect was to severely inhibit red cell production. Higher doses also indicated interaction. Multiple regression analysis was used to study the contributions of the components of the binary mixtures. The results suggestedthat in inhibiting red cell production, muconaldehyde and hydroquinone react at the same site. Muconaldehyde andp-benzoquinoneappear to act at two sites. The nature of these sites are not yet known, but these studies suggest that benzene toxicity is the result of the interaction between benzene metabolites rather than the effect of a single metabolite acting independently. The effects of benzene metabolites on the microenvironment have also beenstudied. Gaido and Wierda (%I-92) evaluated the 298 SNYDER ET&. effects of benzene metabolites on the ability of adherent cells from mouse bone marrow to supportthegmwth of GM-CFU-C cells. They reported that phenol was not effective below millimolar concentrations, whereas hydroquinone and benzoquinone were the most toxic. Catechol and 1,2,4-benzenetriol were less potent than hydroquinone and p-benzoquinone. Previous studies that showed that D W 2 n mice were more sensitive to benzene toxicity than C57/B16mice (93)led Gaido and Wierda (W92t)o examine the relativeability of adherentcells from B6C3F, and DBARJ mice to support hematopoiesis. Although no strain differenceswere observed related to this activity, phenol, but not benzene, reduced the ability of stromal cells to support the development of granulocytdmacrophage precursor cells. Stromal macrophages appear to be a significant target in benzene toxicity. For example,Post et al. (94)foundperoxidasemediated metabolism of phenol to covalently binding species along with theinhibitionof RNA synthesis by hydroquinoneand p-benzoquinone. Renz and Kalf (95)suggestedthat in thesecells hydroquinoneinhibitsthe activationof interleukin-1and the administration of recombinant interleukin-labefore benzene treatment preventedbenzene toxicity in mice. Thesedata suggestthat notonly are severalbenzene metabolitesinvolved in benzene toxicity, but there may also be several targets. It has been demonstrated that stem cells, progenitor cells, and some stromal cells are sensitiveto benzene metabolites. Genotoxicity Mutagenicity Benzene has not been found to produce mutagenicity in shortterm tests in virro (42). The Ames Salmonella test, yeast mutagenicity test, unscheduled DNA synthesis, mouse lymphoma test, 6-thioguanineresistancetest in Chinesehamster V79 cells, the ouabain resistance test in Chinese hamster ovary (CHO) cells, and human lymphoblast HGPRTand TK testsdid not indicatethat benzene was a mutagen. Tests aimed at detec- ting neoplastic cell transformation such as the BALB/c 3T3 mouse fibroblast test, C3H Ull/2 mousecell test, CHOtest, and enhancement of viral (SA79) transformation test were also negative. There was a report that benzene yielded a positive Ames test using strain TAlOO and Aroclor-induced rat liver microsomes, taking advantage of a highly sensitive microflocculation assay (43,%).Another report suggested a positive cell transformation result in Syrian hamster embryo cells (97). The results suggest that except for these two systems, eitherthose cellulartargets that would have to be affectedto yield a positive resultwere not sensitive to benzene, or the negative results were caused by the in- ability of thosesystemstoeffectivelymetabolizebenzene to reactive intermediates capable of producing a positive response.The hepatic stageof benzene metabolismin viw is mediated primarily by cytochrome P-450 IIEl, although under some circumstancescytochrome-P-450IIBl or other cytochromesP-450 may metabolize benzene. Further metabolic activationin bone marrow cells requires peroxidatic activity. In the absence of both types of activatingenzymes, mutagenic metabolites will not be formed and the results would suggestthat exposuE to benzene would not yield a mutagenic effect. Mutagenicity of benzene metabolites, however,has been well demonstrated.Thus, Glatt et al. (43)studied the mutagenicactivity of 13 benzene metabolites using strain TA1535. In the absence of S9, rmns-1,2-benzenedihydrodioland benzene diol epoxides were mutagenic. Dihydrodiol dehydrogenase, which converts the dihydrodiol to catechol, protected against the mutagenic e f k t . It is the postulated, but not demonstrated,that thedihydrodiolcan be convertedtoa seriesof isomericdiol epoxides via a further epoxidation. The anti-diol epoxide was a mutagen in strains TA97, TA98, TA100, TA102, TA104, and TA1535. In V79 cells, the antidiol epoxidealso induced sister chromatidexchange,acquisitionof 6-thioguanineresistanceand ouabainresistance,and micronuclei. Although theantidiol epoxidehasnot beendemonstratedtobeametabolicproductofbenzene asyet,thepotentialforitsformation remains, and itmightbeone of the most mutagenicproducts derived from benzene. Other metabolites effective in the V79 cells were 1,2,4-tri- hydroxybenzene, hydroquinone, catechol, and the r m - l , 2 d i hydrodiol which elevated resistance to 6-thioguanine.The most striking effect in these studies, huwever, was both thepotency and great effectivenessofp-benzoquinonein increasingthe frequency of 6-thioguanineresistance while having no effecton ouabain resistance, and producing no chromosomedamagein V79 cells. Furthermore, p-benzoquinone was relatively ineffective in Salmonella. The potential genotoxic activity of rmns,rruns-muconaldehyde, a chemically reactive ring opening product of benzene in microsomalpreparations,was investigated in studiesby Witz and co-workers (44.45). Tms,rruns-muconaldehyde is generally highly toxic to cells in vim.It is an active mutagenin V79 cells but is weakly mutagenic in Salmonella (44). In CHO cells rm,rmns-muconaldehyde is a potent clastogen (37,45).Thus, it appearsthat if benzene canundergo metabolicactivation,some of its metabolites, both ring closed and ring opened,can mediate mutagenic events. Chromosome Damage Since the first reports of Pollini and co-workers (98,99), benzene hasbeen known to produce chromosomedamage. They cultured both bone marrow cells and peripheral lymphocytes from patientsdisplaying severe benzene hemopathy and reported a high rate of aneuploid cell production. Subsequently, there were many reportedobservationsof chromosomeaberrationsin blood from workers exposed to benzene. Notableamong these were the reports of Forni and her collaborators (60,61),who observedchromosomeaberrationsin ap u p of workersexposed to benzene ata greater frequency thanwrlcers exposedto toluene (60).Follow-up studiesofthese w r k m revealed that bothstable and unstable benzene-inducedchromosomeabemtions occurred. The unstable variety eventually disappear, whereas the stable form persist and may relate to the eventual development of leukemia. Erexson et al. (ZOO) reported that low-dose exposure to benzene (10-28 ppm) over4-6 hr p r o d u d sisterchromatid exchanges (SCE)in peripheral mouse B-lymphocytes. Tice et al. (63)demonstratedthe production of sisterchromatid exchanges in mice after short-term exposure to high concentrations of benzene. The effect was exacerbated when the mice were pre- TOXICOLOGYOF BENZENE 299 treated with phenobarbital, presumably because at these high doses phenobarbital induces an enzyme that can efficiently bioactivate benzene. Although partial hepatectomywas not protective. co-administrationof toluene reduced SCE by 90%.Witz et al. (45)reported that administration of rrans,rmns-muconaldehyde caused SCE in B6C3F, mice. The data suggested that benzene metabolitesplay a significant role in producingSCE. In cultured human lymphocytes in vitro, it is clear that benzene metabolites induce SCE (101). Phenol, catechol, and hydroquinone produced SCE in a dose-dependent manner in the absence of added metabolic activation, and the effect was prevented by glutathione. Sister chromatid exchanges were in- duced with the following order of potencies: catechol > 1,4benzoquinone > hydroquinone, 1,2,4-benzenetriol > phenol > benzene. The report by Hite et al. (62)that benzene produced micro- nuclei in bone marrow polychromaticerythrocytesin mice led to the development of an assay featuring the use of micronuclei in peripheral red cells as a rapid screenfor chromosomaldamage (102) caused by benzene. Since then micronuclei have been observed after benzene administrationvia various doses, routes, and treatment regimens ( 5 ) .Harper et al. (103)suggested that benzene metabolism was necessary to yield micronuclei. Pirozzi et at. (104)demonstratedthat indomethacin,which inhibits the metabolic activation of phenolic metabolites in bone marrow by prostaglandin H synthetase (105).also protects against micronucleus formation. Covalent Binding to Macromolecules The observations that in the course of its metabolism some benzene is converted to reactive metabolites that covalently bind to protein (106,107)RNA and DNA (108)have led to a series of studies on the mechanism of benzene toxicity. Irons and Neptun (109)demonstrated that hydroquinone,but not phenol or catechol, inhibited polymerization of tubulin. Tubulin possesses nucleophilic sulfhydryl groups that bind guanosine triphosphate (GTP), which in turn stabilizestubulin for further polymerization to microtubulesnecessary for spindle formation during cell division (72).Hydroquinone and p benzoquinone interfere with GTP-tubulin binding by alkylating SHgroups (109.110).Another protein that is inhibited by benzene metabolites, hydroquinone,andp-benzoquinone, but not phenol or catechol, is mitochondrial DNA polymerase-y, which also possessesa sulfhydrylgroupat the active site (111).The binding of benzene metabolites to these critical proteins may play a role in benzene toxicity. The observations of chromosome damage described above suggest that the binding of benzene metabolitesto DNA may also represent a significant event in benzene toxicity in view of the chromosomedamage discussedabove. Lutz and Schlatter(108) exposed rats to either ['*C]- or ['Hlbenzene by inhalation and found that liver nuclear DNA contained covalently bound benzene residues, although Lutz (112)notedthat when compared with other carcinogens, relatively little radioactivity derived from benzene was bound to DNA. Rushmore et al. (113)took advantage of the propertiesof mitochondrial DNA to study DNA binding by metabolites of benzene. The mitochondrion contains no mechanism for DNA repair and contains a small circular DNA. Furthermore,the presence of a cytochrome P-450 mono- oxygenase in mitochondria capable of metabolizing benzene facilitated production of reactive intermediates (114). Seven potential DNA adducts on deoxyguanosine and one on deoxyadenosine were observed, and structures have been proposed for one of the adducts on deoxyguanosine (40)and for the deoxyadenosine adduct (115).Neither thymine nor cytosine adducts were investigated. Schlosser et al. (105)investigated the oxidation of hydroquinone by prostaglandin H synthetaseand found that whereas l,Cbenzoquinone, the oxidationproduct of hydroquinone, was bound to cysteine or DNA, inhibition of the enzyme by indomethacin inhibited both types of binding as well as micronucleusformation(104)and benzene-induced myelotoxicity (116). The low level of binding has made it difficult to detect DNA adducts in vivo. However, use has been made of the 3ZP-postlabeling technique to measure benzene related adducts because of its great sensitivity.Bauer et al. (117)reported on DNA adduct formation in rabbit liver. Snyderet al. (87)suggested that DNA adducts are formed in the bone marrowof benzene-treated rats. Using the same method, Reddy et al. (118)was unable to detect DNA adductsfrom benzene in the Zymbal gland, atarget for the carcinogenic activity of benzene in the rat, or in bone marrow, presumably the target for leukemogenic activity. Further research is needed to determine the role of DNA binding of benzene metabolites in benzene toxicity and carcinogenesis. Carcinogenesis From the mechanistic point of view, benzene-induced carcinogeniceventshave been difficult to study because of the lack of a convenient animal model. Chronicexposureof rats and mice to benzene by inhalation (119)suggeststhat benzene might induce acute and chronic myelogenous leukemia and thymic lymphoma in these species. Large scale, chronicexposurestudiesby Maltoni et al. (66,67)in rats and mice using inhalation or oral administration revealed the appearanceof Zymbal gland tumors as well as a variety of other cancers. These results were confirmed and extended in a National Toxicology Program study (68). However, these studieshave not led to the development of models of use to study the mechanism of leukemogenesis. A more pro- mising approach came from Cronkiteet al. (DO),who reason- ed that the difficulty in producing leukemiaduring chronic exposure is because benzene is an inhibitor of cell replication, a process essential to the development of leukemia. Therefore, these workers treated mice for 16weeks with 300ppm of benzene and then ceased exposure. Subsequently, none of 88control mice demonstrated neoplasms, but 8 of 90 benzene-exposed mice displayed lymphoma/leukemias (120).Thismethod has yet to be fullyexploited in the studyof the mechanism of benzene-induced leukemia. Prevailing theories suggest that initiating events in carcinogenesis may be covalent binding of reactive intermediatesto DNA or oxidative damage of DNA. Subsequent attempts to either repair or replicatethe DNA propel the cascadeof events termed carcinogenesis. Furtheranceof this process is thought 10 require the intervention of one or more pfOmOtioml steps that stimulate proliferation of the genetically altered cells. Alternatively, =-a11epi4geneticevents may kid to caEifWems's. The mechanism of benzene toxicity has )'et 10beddhCd these frameworks. I 300 SNYDER ETAL. If we assume that a genetic alterationunderliesthe mechanism of benzene-induced carcinogenesis, we can look toward the results of the genotoxicity studies cited above as indicatorsof the pathway toward neoplasia. Thus. metabolism to reactive intermediates would have to be postulated as an important early step in the mechanism. It would be expected that benzene should then stimulate DNA repair, which would be detectable by unscheduledDNA synthesis. However, when measured in hepatocytes and HeLacells. it did not appear likely that benzene caused DNAdamagesubject torepairinmammaliancellsinculNre(42). Subsequentto a geneticchange, it should be possible to measure cell transformation.ThepositiveSyrianhamsterembryotestsuggested that given the proper metabolicactivating system,benzene metabolitesmay be capable of inducing cell transformation. The second step would require promotion. The possibility that unmetabolizedbenzene might play a promotional role arose from the observationsby Da Silva et al. (121)that benzene activates membrane protein kinase C, which is also activated by tumor promoters such as phorbol esters. This activity is shared with toluene, which produces neither aplasticanemia nor leukemia. However, whereas toluene might activate protein kinase C, its metabolitesare neither hematotoxic nor carcinogenic. Some attempts have been made to investigate the mechanism of benzene carcinogenesisin other organs. Reddy et al. (122)and Low et al. (123) examined the formation of DNA adducts in Zyinbal gland cells in culture and detected DNA binding. However, adducts were not detected in this organ when the animals were treated in vivo. Of perhaps greater significance, however, were the studies of Busby et al. (124),who treated newborn mice with benzene, benzene oxide, racemates of benzene dihydrodiol, and benzene diol epoxide-1 and benzene diol epoxide-2. Although benzene and benzene diol epoxide-1 were inactive in this assay, benzene oxide, the racemates of benzene dihydrodiol, and benzene diol epoxide-2induced lung tumors in the mice. It is likely that benzene oxide and the dihydrodiol were metabolized to the diol epoxide-2 to initiate these tumors. It will be importantto evaluate the likelihood that the diol epoxidecan be formed in bone marrow in vivo to deter- mine whether it can be important in leukemogenesis. One mechanism that might explainthe initiationof leukemogenesis by benzene derives from recent considerationsrelating chromosomeaberrationswith cancer (125).Although chromosome breaks and sister chromatidexchanges have been identified as outcomes of benzene exposure, specific studies of the possibilities of chromosometranslocationshave not been performed. Metcalf et al. (126)discussed the relationship between translocationsand leukemogenesisand recalled that about two dozen nonrandom chromosome translocations have been reported in both B-cell and T-cell tumors. Often these translocations have resulted in the close association of a proto-oncogene with a gene that codes for an immunoglobulin in a Rlymphocyte or a T-cell receptor in a T-lymphocyte. The proto-oncogene is then deregulated and can play an important role leadingto the expansion a neoplasticclone. A recent example of thisphenomenon w a s reported by de The et ai. (127),who demonstratedan association of a specific translocation, designated as t(15;17), with acute promyelocytic leukemia, in which the net effect is the fusion of the retinoic acid receptor, RARa,with a gene called PML.These authors suggest that the translocation results in the formationof an RAR mutant, the activity of which is to interfere with promyelocytic differentiationand thereby facilitatethe pmcess of leukemogenesis. Thus, at least three types of chromosome changes caused by benzene metabolites, i.e., gross chromosome aberrations,micronuclei, and sister chromatid exchange, have been reported. If benzene metabolites can produce translocationsas well, this might be a mechanism for leukemogenesis that is parallel to the example cited above. Pharmacokineticsof Benzene There have been a number of attemptsto developpharmacokineticmodels of the fateof benzene with the intention of applying the results to risk assessments for benzene. The first attempt to model the fate of benzene was made by Sat0 et al. (128)who exposed three men to a single exposure of 25 or 100 ppm of benzene for 2 hr and then observed a triexponential decay of benzene from their blood. Sat0 et al. constructed a threecompartment model made up of richly perfused tissues, poorly perfused tissues, and fat, which acted as a major sink for benzene. Snyder et al. (18)repetitively exposed mice or rats to 100or 300ppm of benzenefor 20 daysand sampledthe blood for benzene on days I, 6, and 20. At higher doses mice achieved greater increases in blood levels of benzene than the rats. The mice alsodisplayeda greater elimination rate constant than the rats. The rate of blood benzene disappearance from mice at the high dose shifted from monoexponential to biexponential between the 6th and 20th day, which may have been due to enzyme induction ofbenzene hydroxylase, i.e., cytochromeP-450 DEI. More recent approacheshave attempted to factor metabolism intophysiologicallybased pharmacokinetic models of the fateof benzene and to developa method for extrapolating from animal models to humans. Belilesand Totman(129)constructeda model using Zymbal gland tumors and blood related cancers as end points and, recognizing thepossibilitiesof synergyand multiple mechanisms, assumed that the combined metabolites represented the "reactive agent." Using largely the data of Sabourin et al. (13) and applying an allometric technique to estimate scaling between species, they suggested that their model, which predicts that a working lifetime exposure (40-45 years) to benzene at 10 ppm would result in 6-14 cases of Ieukemid1000,is in good agreement with federal regulatory risk assessments based on epidemiologicaldata. Physiologically based pharmacokinetic models aimed at relating benzene metabolism and interspeciesdifferences,using both experimental data and simulations, have been developed by Medinsky et al. (130),Travis et al. (131,132),Boiset al. (133), and Spear et al. (134). Each described a multicompartment system and attempted to relate end points to the generation of metabolites with emphasison specificmetabolitesassumed to be responsible for generating the end points under consideration. Thus, Medinsky et al. (130)considered the role of hydroquinone and muconaldehydegenerated in the liver and Bois et al. (133) added the metabolic activity of the bone marrow in the further metabolism of metabolites generated in liver. While each representsan important advance in interpretingthe metabolic fate of benzene in relation to the development of benzene-related disease, they all suffer from lack of sufficient data, as well as from variability of the databetween experimentaldesigns. There generally is difficulty in fitting empirical data to theoretically .- TOXICOLOGYOF BENZENE 30 I generated curves, which suggests that the models need to be improved. Whereas thedisappearance of benzene from the blood, following administration via any route, can be measured readily, the requirement for metabolism before benzene can produce serious chronic effects, makes modelingdifficult because of the uncertainties pertaining to which metabolites are responsible, the relative impact of each, and the potential for multiple target effects. A large number of metabolites have been described above. Because of their demonstrated, direct effects on bone marrow, pharmacokinetic models have concentrated on hydroquinone, alone or in combinationwith phenol, and muconaldehyde, and for the most part have assumed that they are formed in liver and transported to the bone marrow. The possibilitythat someof the critical metabolites are ultimately formed in bone marrow serves to complicate the modeling process. For example,although the metabolitestransferred to the bone marrow may be innocuousin themselves, further metabolic activation in bone marrow, perhaps mediated by peroxidases, may generatethe ultimate toxic metabolites(135).Thus, hydroquinone may be released from the liver, free or conjugated, reach the bone marrow by some mechanism, and in its free form may be oxidized to p-benzoquinone by a peroxidase. Muconaldehyde might be formed in bone marrow, and the two working in concert would then produce a toxic effect. The models would have to reflect these factors, as well as take into account the activity of other potential toxic metabolites, if they are to accurately picture the processand be used in risk assessment with accuracy. Human Impact Benzene is a ubiquitous pollutant in both the workplace and in the general environment. Ambient concentrations can be detected worldwide, including pristine areas, although at levels much lower than in modern urban societies. Major sources of benzene include petroleum and petroleum products. Levels of benzene in gasoline range from approximately 1 to 5%. Gasoline serves as a major source of benzene exposure in a wide variety of situationsincluding groundwatercontaminationthrough leaky underground storage tanks; air contamination through the evaporation of gasoline in workplaces and in homes; and transdermal exposure when gasolinegets on the skin. The major cause of high-level exposure remains inappropriateuse in an unregulated workplace; although control measuresare leading to a decreased use of benzeneas a solvent and organic synthetic starting block. A major source of personal benzene exposure comes from cigarette smoking(136),and benzene is alsopresent in sidestream smokeleading to exposure through passive smoking. Although there are measurable amounts of benzene in certain foods, these are no longer believed to be a significantsource of benzene exposureto the general public, particularly in comparison to indoor air pollution from gasoline and benzenecontaining solventsor from cigarettesmoke within the home or the office (137). Nonhematological Effects The odor threshold for benzene is in the range of 4-5 ppm (138).The acute nervous system toxicity of benzene appears similar to the general anestheticeffectsof lipophilicsolventsand is assumed to be a direct effect of benzene unrelated to its metabolites. Acute symptomsincludedrowsiness, lightheadedness, headache, delirium, vertigo, and narcosis. Levels at which acutecentral nervous systemeffectsbecome apparentare at least above 100 ppm, an earlier occupational health standard for benzene, although milder effects could conceivably occur at lower levels. Based on structure-activity relationships,benzene might be expected to have acute central nervous system effects similar to alkyl benzenes at a slightly lower dose for benzene. Chronic newus system effects of benzene have not been clearly demonstrated. Nonhematological tumors have also been demonstrated in long-term animal studies(139,140).However, at present there is no reasonably convincing evidence of nonhematological solid tumors occurring in humans exposed to benzene. A metaanalysis of benzene-exposed populationsmay be of value in this regard. Studiesof the productiveand developmental effects of benzene have shown little aside from hematological effects in the fetus (7778).A more thorough evaluation has been called for by Davis and Pope (141). Immune dysfunction as a result of high-level exposure to benzene in laboratory animals has been clearly demonstrated (142).Lymphocytopeniais an early effect of benzene exposure in animals and in man (143), and there is also evidence suggesting that the immune system can be affected by benzene independent of lymphocytopenia(144).However, there is currently no evidenceto demonstrate an effect on the immune system of humans exposed to allowablelevels of benzene. Hematological Effects In humans, as in animal models, the bone marrow is the target of benzene toxicity. A major unansweredquestion about benzene toxicity concernsits primary localizationto the bone marrow, a finding that isalmost unique in solventtoxicology. As with many other solvents, benzene is primarily metabolized in the liver, yet despite evidence that metabolism is necessary for bone marrow toxicity,hepatotoxicity is not observed. In essence, there are two different explanations given for bone marrow localization: specificity in the local metabolism of benzene within the bone marrow or particularities in the susceptibility of the bone marrow, as compared to the liver or other organ, to a benzene metabolite or metabolites. For example, Subrahmanyam et al. (135)recently emphasized the potential role of free radicals in benzene toxicity, pointing out that the relatively high level of myeloperoxidase and of other peroxidasesmay be responsible for localizationof benzene toxicity to the bone marrow. In contrast, Goldstein and Witz (145),noting the relative sensitivity of the bone marrow to chemotherapeuticalkylating agents, have focused on a potential hematologic role for truns,rmns-muconaldehyde, an cY,&unsaturated aldehyde alkylating agent which, although highly reactive, has a sufficienthalf-life to theoretically travel from liver to bone marrow. Benzene was initially identified as being a human hematological toxin in the 19thcentury. Sincethat time the literature is replete with case studiesand series in which fatal aplastic anemia has been reported in individuals exposed to significant levelsof benzene (146,147). As described above, aplastic anemia is a . I 302 SNYDER ETAL.. serious, often fatal, disorder in which the formed elementsof the bone marrow are replaced by fat and there is a significantdecrement in the formed elementsof the blood. Humansmay display a decrease in white bloodcells (leukopenia),potentially resulting in death due to infection; a decrease in platelet a u n t (thrombocytopenia), potentially resulting in death due to hemorrhage; and a decrease in red blood cell count (anemia). A lesser extent of benzene exposure produces lesser degrees of damage to the bone marrow. The normal human bone marrow has sufficient reserve capacity to be able to produce approximately six times more red blood cells under stress than it does under normal conditions. There is also a wide range of statisticallynormal values in peripheral blood counts. Thus, earliest formsof bone marrow damagedue to a compound such as benzene may not be notable in terms of adecrease in circulating blood counts. As described above, studies in laboratory animals have shown that a decline in colony forming cells indicative of the function and number of bone marrow precursors can be seen with relatively low levels of benzene exposure. In humans, individuals with benzene exposure may initially have acount lower than statisticallynormal in any one of the formedelements, but with significantexposure, pancytopenia will be observed (146). There is a wide range between thoseblood count levels which are statisticallyabnormally low and those which produce functional abnormalities. Thus, the rangeof normal in a laboratory platelet count is often 150,000-350,000/pL; yet evidenceof abnormal hemostasis is usually not observed in humans until the platelet count is less than 50,000/pL. There is, however, some evidence that in significant benzene toxicity not only is there a quantitativedecrease in formed blood cell elements, but there is also a qualitative alteration. Such qualitative changes might be expected to interfere with function of platelets or white blood cells. A hallmark of benzene effect in red cells is an increase in average cell size, known as the mean corpuscular volume (calculated by dividing the hematocrit by the red blood cell count). An increasein the mean corpuscularvolume alsooccurs with folic acid deficiency and vitamin B,2 deficiency, two disorders which have in mmmon an interferencewith DNA synthesis in the bone marrow. An increase in the mean corpuscular volume is observed in almost any form of aplasticanemia. As an increased mean corpuscular volume also occurs relatively early in alcoholism, as well as other conditions, it cannot be taken as a definitive test for benzene exposure at the workplace, although it can be a useful confirmatory clue (143). As with any toxicant, there appears to be a variation in the extent to which individualsdiffer in their sensitivitytobenzene, and some suggestionof a familial tendency (148).However, there is no evidenceof an idiosyncratic low-level sensitivityto benzeneinduced aplasticanemiasimilar to that observedwith chloramphenicol. Interaction with other agentsmay affectbenzene toxicity as described above with respect to ethanol and toluene. Metabolic interaction between tolueneand benzene would be expected when exposureoccursto a mixture containinga high ratio of toluenehenzene becausetoluene isa competitiveinhibitorof benzene metabolism (1).However, Sat0 and Nakajima (128) reported that there is no significant interaction betwen benzene and toluene with respect to their fate when human experimental exposure was near the threshold limit value. Neoplastic Effects LeNoir and Claude reported the first case of benzeneassociated leukemia in humans in 1897 (149)but the diagnosis was not well documented. In 1965 Browning (149)recorded 61 cases, and by 1977 Goldstein (146)had assembled 121cases of benzene associated leukemia. The studiesof Vigliani et id.(IN), Akmy et al. (151),and I n b t e et al. (IS?),each of which examin- ed large populations, presented convincing evidence of the leukemogenicactivity of benzene. There is unequivocal evidence that benzene is a causeof acute myelogenous leukemia (AML), the adult form of acute leukemia. Individualcases in benzene-exposed individualsbegan to be reported in the 192Os, but the causal relation was not fully accepted until less than two decades ago. This relationshipwas first accepted by hematologists who have long recognized that anyone with aplastic anemia from apparently any cause has an increased risk of AML. For example, AML as a second tumor is, unfortunately, common in cancer patients whose bone marrow is affected by radiation and alkylating agents used in chemotherapy(153).Benzene exposurealso leads to cytogenetic abnormalities in bone marrow cells and in circulating lymphocytes (60,61). Epidemiological evidence strongly supportive of the causal relationship between benzene exposure in the workplace and acute myelogenousleukemia includesstudiesof shoe and other leather workersin Italy and in Turkey (1NJ54).In Turkey, a glue used in the fashioningof leather goods which had relatively low levelsof benzene as a solvent was replaced in the 1960swith one containing much higher levels. Soon thereafter, Aksoy and his colleagues reported a wave of patients with aplastic anemia followed by identificationof numerous individualswith AML, many of whom had previously been identifiedas having aplastic anemia (154,155).Studiesof large cohorts of benzene-exposed workers in thechemical and petrochemical industrieshave tended to show an increase in hematological neoplasms (156-160). The most thoroughly studied cohort has come from Goodyear Rubber facilities in Ohio. Repetitive follow-up of a cohort of workers by the National Institute of Occupational Safety and Health (NIOSH) has identified at least 10 cases of acute and chronic myelogenous leukemia, with only two expected (152,161,262)T.o obtaina better understanding of the level and pattern of benzene exposure associated with this increased risk of AML, NIOSH has performed one of the most thorough retrospective exposure assessments ever done on a cohort of workers (162).This exposureanalysis has been used as a basis for the current risk assessment of benzene by the U.S.En- vironmental ProtectionAgency (EPA). However, it does appear that NIOSH has significantly underestimatedthe extent of exposure in the Goodyear plants during World War I1 (163,164, thus leading to some overestimation of the risk of benzene derived fromthis cohort. However, it should be emphasized that the current ERA risk assessment of benzene hasmore uncertainty associated with the dose response part of this equation than it does with the exposure. Despite the many argumentsconcerning the extent to which wrkers who developed AML were exposed to benzene, the differences all lie within an order of magnitude. In contrast, thedose-responserelationshipbetween daily environmentalbenzene exposure in the range of parts per billion and leukemia has a much larger degree of uncertainty. TOXICOLOGY OF BENZENE 303 This uncertainty can only be resolved by a better mechanistic understandingof benzene leukemogenesis. More problematicis the potential causal relationship between benzene and lymphopoietic cancers in humans. While in no case is the evidence incontrovertible, there does appear to be more than sufficient grounds to link benzene exposurewith multiple myeloma (165)and perhaps with certain forms of non-Hodgkin's lymphoma and acute lymphoblastic leukemia. In the case of multiple myeloma the observations include individual case reports and a statistically significantly increased number of myeloma cases in the Pliofilm cohort that is under study by NIOSH (162,166)I.n the latter cohort, the association has been suggested to be problematic in view of the lack of relationship with the extentof exposure. However,as there is a small number of cases, it is difficult to expect an obvious dose relationship. There is a strongelementof biologicalplausibilitythat supports a causal relationship between benzene exposure and multiple myeloma, as well as other lymphatic neoplasms. Lymphocytes are definitely a target of benzene, with lymphocytedepletionoccurring early in the course of benzene exposure(146,167).The readily detected cytogenetic abnormalities in lymphocytes following significantbemne exposureclearly indicate that lymphocyte DNA is affected as a result of benzene exposure, and there is no question that hematopoietic tissue is subjected to a carcinogenic metabolite of benzene in view of the clear causal relationship with acute myelogenous leukemia. Non-Hodgkin'slymphomas represent a grab bag of different diseases. A certain portion of these disordersreflects severe immune depletion as occurs with AIDS or secondary to treatment related to organ transplantation, in which the tumors occur very rapidly, sometimes in less than 1 year. However, there is no clear evidence that benzene produces this fbrm of immunedepletion. Non-Hodgkin'slymphoma is observed in certain work groups that have been exposed to solvents or other chemicals, but the evidence that benzene is causally related remains suggestivebut uncertain. Similararguments as to biomedical plausibility could be used to link benzene exposure to acute lymphoblastic leukemia, the leukemia most commonly observed in children. However. in view of the fortunate absence of well-defined populations of children that have been exposed to benzene, it will be particularly difficult to develop the epidemiological data necessary to evaluate the potential causal relation. Individual cases in relationship to household benzene exposure are being observed medically and legally but have not yet been reported in the clinical literature. REFERENCES 1. Andrews. L. S . , Lee, E. W.. Witmer, C. M., Kocsis, J. J., and Snyder, R. Effectsof toluene on the metabolism. disposition,and hemopoietic toxicity of 'H benzene. Biochem. Pharmacol. 26: 293-300 (1977). 2. Sammdt. 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