Document 5LOy2JbjBkvDa0QwEEv1g32RN

7-f 77- / I CURRENT CONCEPTS OF CHRONIC BEh'ZENF TOXIC`ITI' / Authors, Robert Snyder James J. Kocsis Department of Pharmacoloey Thoma s J eff er s o n L'nI ve r SIIy Philadelphia, Pennsylvania Referee Roherf Drew National I n r i i i u t r o f t nvirr,nmentsl Health Sciences Research l r u n s l r Park. North Carolane ;c`- il INTRODUCTION The maladies that have beset mankind throughout history may in general be classified as parasitic ( i n f e c t i o n s, in festations, etc.), nutritional/ endocrine disease (hyper. or hypofunctional e n docrine organs, etc.), or traumatic injury (accidents, war, etc.). With the development of an industrial society, new forms o f disease arose in which people succumbed to illness induced by exposure to toxic materials in the course of their *'labor. Occupational diseases have been with us for m y centuries.' but the incidence of indus. ,:.I toxicity increased markedly with the advent of the industrial revolution. Although efforts to protect workers against jobrelated illness have been vigorous in recent years, economic necessity compels the continued UK of hazardous chemicals, and benirne is an excellent cmw in point. It hms been estimated by the National Institute for Occupa- tional Safety and Health that about 2,000000 perrons in the national work force have potential exposure to benzene. In 1922 Alice Hamilton,' the noted student of indust rial toxicology, reported on "The Growing Menace of Benzene (Benzol) Pobonins in American Industry." She W L ~familiar with the d e r r i p - tions of benzene toxicity reported by Santenon` rnd and she reviewed the incidence of ----I b-oth -a..c-u--t-e -an-d chronic toxicity with t h e intention of aJertinR the medical profession and i n d u S t r v 3 tlle hazards of benzene. During the next several er- taken to restrict benzene utilization, and in 1978 it was ponible for her to write a foUow-up paper, "The Lessening Menace of Benzol Poisoning in American Industry.'" Despite the optimism generated by the title, she reported that benzene utilization remained heavy in industries concerned with the manufacture of rubber, artificial leather, cans, paints, certain artificial furs. and in dry cleaning. With the advent of high-speed printing presses in the 1930s and the consequent need for fast-dning inks. benzene was again pressed into service because it was ancxcrHcnt. &t for ink and it evaporated rapidly. Thus, despite atfempts to decrease overall utilization ofa benzene both in this country and abroad, by the middle 1930s there remained. unfortunately, an abundance of clinical material in which to studv bcn zene toxicity. The development of the chemical industry during World War 11 and in the postwar era. especially in the field of plastics, f m l y reestab. lirhed the necessity for large quantities of benzene June 1975 26s will nrubahh remain with us as long as the Gnsequence to the medical communlty is that we must continue our efforts to gain a complete understanding of benzene toxicity if we are to learn how to prevent o r to deal with the illness resulting from exposure to bcnrrne. The most serious effect of chronic cxmsure ty bcnizne is depression of the-b row.' The effect is unique among aromatic hydrocarbons and is not shared by simple alkyl derivatives o f benzene.' ' Acute exposure to high concentrations of beniene in the atmosphere has led to loa of consciousness since benzene ;long with many other hydrocarbons exerts a depressant effect o n the central nervous system that appears to be related to its thermodynamic activity in aqueous solution.' " Both acute and subacute e x p a u r e to bcnzrne has resulted in hisrochernical changes in 'kltlrlc\ ' .-I irt1-r : sninll .c infc5iiiie.- 2nd 'bpiriJl ,(lid IIic hcar1 is also affected.' - atid it lias hvcii rcportrd that indi\iJu;lls pcri~i~riniiig et rviiuou< pti! ~ i c la~htor are especidll) cusieprible t t i heii/e.ric t o \ i c i t \ ! I ~ i n i l ~ o n 'd"e s i r t h e c I CIIII:!- 1 i i i r i I I I u t i i ~ l i3 pcrcon entered a taiih previou4h used III s i o r c henieric. w3c overconie h! tlic. fumes. and l ( ~ tconsciousness A co-wui her u t i 0 entered [he tanh Ui11iout rrqvratorl protectiori was himself overc'ome while attempting to rescue the unc'onciious person and died. The first 6 I c t i r n was subsequerirl) removed from the tank b! adequately protected personnel. regamed con- sci(uIcriess. and sunived. Browning' summsrized the effects on the heart by indicating that during stressful ~ituationcexcessive carecholanitne< arc pruduced hy the adrenal and that bcnrerie Itkc some other general anesthetic agrnts such as cyclopropane and the halogenated anesthetics sen- siti7es the heart to catecholarninc~.'~The result may he ventricular fibrillation foilowed by desth Thus, it may he surmised that the first worker lost consciousness due to the central nervous syslein depressant activity of benzene and regained coli- sciousness when removed from the benzene-rich ~tmosphere whereas his would-be rescuer suc- cumbed to the cardiotoxic activity of benzene while struggling to save his companion. The incidence of acute toxicity is iou hut in stressful situations may be fatal The symptoms of' acute and chronic benzene toxicity are different. and while benzene itself appears to cause acute toxicity. considerable study has gone into evaluating the role of brnzene metabolites as etiologic factors in chronic toxicity. Therefore, because acute benzene toxicity is of sufficient interest and complexity to generate a separate discussion. this review will concentrate on chronic benzene toxi. CIt} The opening sectioii will describe chronic hew zene toxicity in humans and will be followed b) a discussion of reports on the mechanism of benzene toxicity derived largely from animal studiec. The discussion will continue with a review of current thought on whether benzene toxicity may lead tu leukemia and o n immunological effecfs of hen. zene. The next two sections deal with benzene metabolism in vivo and in vitro because there is a strong pmihility that a metabolite of heriiene rather than hcnztne itself i s responsible f u r ben=ne toxicity. Thr last section reviews studies specifically aimed at attempting to relate benzene mtabolism to benzene toxicity. CHRONIC BENZENE TOXICITY IN HUMANS The earliest reports of benzene toamt)'-6 descnhe the developmen! of aplastic anemia as a of chronic exposure to bcnzene. DCSPIICmany years of study, our understanding of the effects of benzene on bone marrow is limited in part because the structure and function of the bone marrow habe not been well understood and in part because the concept of aplastic anemia has undergone evolution s i n a the first descriptions of benzene toxicity s,30-34 In normal adul t s blood cell format ion occurs in the marrow of the central bones and the proximal ends of the femur and h ~ m e r u s . ~ 'The totd volume of the marrow cavity equals about 30 to 50 rnllkp body weight of which about half contains active marrow The cells produced in this organ include not only erythrocytes, leukocytes, and thrombocytes but also some types of lymphoc y t e ~ ' I~n adults the marrow usually has sufficient reserve capacity to obviate the necessity for extramedullary hematopoiesis. Early studies of benzene toxicity suggested that benzene crused aplastic anemia resulting in pan- 'cytopenia.' Subsequently pancytopnia was equated with aplastic anemia Later investigation of bone m a m w mean in benzene toxicity revealed cases in which the marrow appeared to be hyperplastic despite pancytopenia of the drcu- 'Iating blood.' q3 If the current definition of aplastic anemia, i.e., pancytopenia accompanied by fatty displacement of bone manow,'' is applied, then pancytopenia accompanied by hyperplastic bone manow is not aplastic m e m u and may be a sign of the preleukemic state or of true leukemia. The posible role of benzene in the induction of leukemia will be d h c u s c d later in this review. In pancytopenia characterized by a hypoplastic bone marrow sternum,b .3 b ,3 0-4 i ribs and vertebrae display marked displncement of red marrow by fat, rnd fibrotic areas in which f i t h i t been replaced by collagen have been d a c n l w d . In some areas ineffective attempts i t intramedullary comp n u t i o n are exemplified by pockets of nucleated red cells. while other pockets contaln phagocytes laden with hemoriderin. Earinophilic urd ncutro- plulic granulocytes may be present in numbers equal to or less t h a n those of erythroid cells. Mcgakaryocytes are largely absent. All of these observations coincide with the finding that throughout the marrow mitotic figgres are infrequent. These observations made either in the late stages of benzene toxicity or during post-mortem examination provide a reasonable basis for the pmcytopcnia of benzene toxicity. Although various stapes of increasing severit) during the progression of benzene toxicity in man have been observed in different individuals. the complete course of the disease cannot be experi. mentally investigated in man because it eventually reaches a stage which is irreversible. Indeed. most the t y p i d picture ot an i n m u a l in the late stages of benzene toxicity is one of general debilitation displaying purpura and bleeding from mucuous membranes.39 Helmer4 reported that among 60 cases of chronic benzene poisoning in various stages, 729, complained of headache, 887 of tiredness, and 44% of cutaneous hemorrhages The detection and description of the early stages of benzene toxicity have been a less frequent Occurrence because in the absence of routine screening programs at places o f employmenti-p; -baet that thev are victim5 afkum.e Doisoning until the disease has become, -m~e . M-ost of the reported cases of the earlier stages of benzene toxicity were detected when a large group of workers were screened as part of a study subsequent to reports o f severe toxicity due to benzene exposure in a specific indust v , w = + b whereas other reports were more concerned with later, more serious4 examples of the disease. The reconstwction of the progress of the disease comes from synthesis of the observations made o n people at various stages of toxicity supplemented with inferences taken from animal studies. In its initial stages benzene toxicity is manifested as a paradoxical alteration of the blood picture. Polycythemia and anemia, leuce cytosis and leucopenia, thrombocytosis and thrombocytopenia have all been reported in the urne studies. With continued exposure. however, the trend is toward decreased levels of circulating erythrocytes, leucocytes. and thrombocytes. As the disease intensifies, circulating blood cell levels decnrse further as pmcytopenia develops. June1975 267 In man decreases in red cell levels are a frequent indicator of early chronic benzene poisoning Among 89 individuals studied by Hunter,44 159 studied by Goldwatcr." and 60 nudied by Hclmcr.42 37. 4R. and 97'7. respcctlvely, displayed eqthroLyte levels belou 4.5 millionimm'. The ancmu IS dncrihed as macrocytic and hyperchromic The furmer IS a frequent consequence of bone marrow depression, and the latter appears to be related to the fact that while there is a decrease in total hemoglobin concentration the reduction in red cell numbers is much greater, the net effect being hyperchromicity. I 6 .I P ,4 I ,S 0 - 5 2 Because benzene toxicity hu been intensively studied through most of this century, the report of a sign or symptom not pre\ioudy described is rare. Thus. it was with consderable interest that investigators in this field read the report of A h o y et al.," who demonstrated that worken in Turkey exposed to benzene and suffering from pan _7 2 -%p i a displayed increased levels of fet giobin_(HbF) inTheir b h d . HbF% a promment Gmoglobin variant - n a l y found in the fetus and at birth constitutes between 50 and 65'7 of total hemoglobin." HbF decreases in early life to below lclc at the age of IO and is not usually observed in normal adult blood. It is usually elevated in the thalassernhs and is often associated with elevated levels of HbA,. mother hemoglobin variant in these d i s e a ~ s . " * ~It~ is important lo recall that thalauemia may be manifested from childhood as a serious anemia called thalassemia major (Coolcy'r Anemia) or m y occur asymptomatically in carriers for the disease. in which case it is called thalassemia minor. An intermediate form of the disease is also known. S M i d i et id." and A h o y and Secer" had peviously demonstrated that HbF is frequently observed in patients suffering from aplastic anemia. and Bloom and Diamond'' showed that among a large group of children in Boston suffering from aplastic anemia with elevated HbF levels, those children in whom the HbF was present in excess of 400 m%;c survived while those with less HbF Jl d i d . A.kmy-et-d." reported t v n g 24 patients with chronic benzeqg t w v and pmcytopcnuSgTiaf.mrr CWlS 8bOW s m f l rnrthe remainder were below that level. All s I t e w e r level survived. sugge- I." tLt the HbF c o n c e s tntion in blood VI not neccmrTy p6gnoCtic f; _- recovery from aplastic a n e m i a .j-n d d t + e h . c Lenl The significance of the relative values of mF levels in predicting SUMMdIuring aplastic anemia may lie in the different populations that were studied. Thalassemia is a disease endemic to Turkey. whereas it is relatively rare in Boston. 'Although A k w y et ai.' showed that HhA2 levtls were normal in 21 out of 24 patients. sugemting that the): were not thalassemic. it has been reported that elevated HbAt is not alu,ays seen in thalassemia s 4 Fu-thermore, the possible role of benzene in modify '8 HbA2 levels has yet to be --inrestigilted. Therefore. it would be of interest to det-erm_ine the various hemonlobin variants in a p o u p of worken similatlv CxDosed to b e n z w who h o w no s i p s of benzene toxicity or have not- develop-cytopcnia. I t mav be- thalassemia can e i t h e w e d i s p o s e or convev profection _i _q-a. b t benzene tuicky. Alternatively, benzene may either mask or exacerbate some of the signs of thalassemia. Further studies in this area would be welcomed by students of benzene toxicity. Although there is some evidence for an increase in red cell hemolysb in benzene t ~ x i c i t y , t' h~e ~ ~ ~ anemia i s most commonly thought t o be the result of decreased red cell production. Detection of the earliest stapes of reduced hemopoiesis is not possible using cell counting techniques because of the long life span of the red cell. If the normal mechanisms for removal of aged or damaged cells . from the circulation are operational, approxi- mately 1/120 of the erythrocyte population is removed and must be replaced per day. I t is likely that early in benzene toxicity erythrocyte produc- tion b reduced but not prevented and some -portion of the red cells can be replaced despite benzene toxicity. The result is that the time tthSnq&a counts may be quite long. Hunter4' reported that by the time exposure to benzene caused anemia, depression of leukocytes and/or thrombocytes was apparent. U u the ~~QGUUU of anemu w sinnifv benzene toxicity of long standing. In contrast, the snorter life span of the leuko- cyte and the fact :hat there cells tend to leave the bloodstream and not return suggest that detecting decreased white cell production should be simpler than detecting a changc in red cells. The picture is complicated by the fact that the white cells are a complex mixture of cell t y p , iome of which d o not arise In bone marrow. k u k O p m l 8 h a been described a the earliest6 and most frequent" sign of benzene toxicity. I n a rtudy of 200 women exposed to benzene in the course of their occupa- tion, neutropenia was reported in 50 cues,a more generalized leukopenia in 44, and anemia in 41.'* The finding of leukopenia in the absence of other bone marrow abnorrnditie*however, is relatively rare ' ? * 4 4 - " White cell values have been reported to drop from levels of 5 , 0 0 0 to 10,OOOullslmm' to below 1,000 in benzene toxicity." Despite the general agreement that benzene toxici6 results in Ihopenia, ettectivc w e e n i n g- for 6rnzene toxi- C r n O U l d not -be m e- lOK8f leicoc e levels only. sd* who suffered from benzene e6toxicity iid n ~ to h e m o n h q e . Both Santelson' and Scl- described excessive bleeding in benzene toxicity, but Duke'" was the first t o describe the reduction in thrombocyte levels. Nikulinr and Titowa6' suggested that thrombocytopenia may be unong the earliest signs of benzolism, and Goldwater" showed that platelet counts were depressed in over 30% of hls rotogravure workers e x p e d to benzene. The observations b y Mitnik and Genkin' and Brocher' 'I that platelet levels may drop from about 250,000 all4 m' to below 10,OOO are an indication of the severity of thrombocytopenia in advanced benzene toxicity. In addition to decreased num- bers of platelets, Saita and Sertoli6' reported that the ability of platelets to aggregate was dtpreued by benzene. Saitr et a!." -sed that there may be several defects in clotting but that both decreased levels of platelets and a decrease in thromboplastic factor activity were important. The time required for t h e e events to occur varies greatly unong individuals. $om w o r k l p hc e e n b w n to h o w ti& of benzene toxicity after brief exposure to relative_lv-low a c e n t r a t i o n s , whereas manv diiolav resis.taue blood eI!s. i 7 n the production of normal ANIMAL STUDIES ON CHRONIC BENZENE TOXICITY The early reports of Santcsyson' and Selling* of benzene toxiaty in humans w e e accompanied by descriptions of experimentally induced benzene toxicity in animals. The obsrrvations of paa; cytolwnia and bone m r r o w depression in anima!j. h i l a r to thore in man IoOowing chronic exposun to benzene, suggested that animal modelswould be useful in the rtudy of the mechanism of - ~ __ - exDoIure and degree of ioxiciiv in variw ? Z K e p o r t s can be chosen to exem relationship. Latta and Davits" admini- stered benzene to rats subcutaneously at doses of 2 to 4 ml/kg/day, whereas Dcichmann et 11.66 d rats to benzene i n h atmosphere and d toxic effects in the ranm of 65 t o 83!, ~ mSel;ling6 gave rabbits benzene subcutaneously at a dose of I ml/kg, while Weiskotten et d6' exposed rabbits to atmospheric benzene at a level which we have d c u l a t e d t o be 240 ppm.. Although an initial transitory leucocytosis was frequently observca. the eventual result in each case regardless of the species or the route -administration - __ was reucopenia. At lower dose_s - %ore t i Z w U required: but the eventual was the same. 1he predominant effect WBS neutro- . penii ac;ompanied by an apparent Ibniphucytosis cytc pruductiun occur qulte early after exposure, w h i i h graduall! disappeared as hcnzenc attacked -I\rnl;huid tissuel(btta and Davits*' rwmt I ~ r n p t ~ a t l ctirsuc was more sensitive to b e n z q but their detection requires methods mwe sensitive than counting the number of circulating cells. The rnechaiiism o f henzene toxicity has been -t h a n r n ) e l ~ ~ i ~ isnuracts, while Sell that -e was true in rahhits periia IS characterized h ) a shift to the left in the studied in hone marrow of experimental animals. The clasical studies o f a h included a descrip tion o f rabbit bone marrow following chronic 4rnftlI count. which SU&CS~S that l e u c o c y t ~ in! oxication with subcut aneuusly administered rnatur3110n IS Impaired. The leucopenia can occu? ell counts may reach extrem-ely 10;~ lelrjLtorie;uh The use o f animal models to determine which cell typ( i s most sensitive to henzenc in order that gradually disappeared during the course of treat- monttorinp of that cell type nian might be used ment. Considerable- to monitor benzene toxicity may in retrospect to supp%csis that benzene produces % have been relatively unprofitahle. In man argu- erfeci-bv inhibrfipp mitosis. Fewer mitotic figures ments haie been advanced to demonstrate that are observed in marrow of benzene-intoxicated each o f !he cell types may be an early indicator of benzene exposure i f its level in circulating blood *animals, and ahnormal mitotic figures have been described by Parmentier and Dustin" and Pollini et al." Chrowosome aberrations follcwmp ben- v- , -zene treatbent or exposure have been reported bsl, %sling and Spcck, " '- i- om1 et ai ," a n 4 L f-o u~p h et ~ ~ - 1 . ' A~lthough cellular damagc which results in mitotic arrest is readily observed. the initial attack may well have been inflicted at any of several stages in the cell cycle. Thus. Moeschlin and Speck" and Kissling and treated to measure and are not commonly used as indica- rahbits with benzene subcutaneously and reported tors of benzene exposure. Therefore. leukocytes inhibition of incorporation of tritiated thymidine which suffer from neither drawback have usually been reported in animal studies as the first cell type to be de?leted. It must be stressed. however. that no conclusive evidence exists to show that into DNA and tritiated cytidine into RNA re- --spectively. They claim that benzene inhibited the synthesis of both types of nucleic acids. Boje et d." exposed rats t o atmospheric benzene until- benzene preferentially depresses the production of any individual cell line in the bone marrow. dgns of benzene toxicity were apparent in peri~ era1 blood and also administered tritiated thymj- In an attempt to detect early effects of benzene 'on red :ell production. we6" *6 devised a method $ne to det.-er_m_.-i-ne t h-e-eff-e-cts o f b e n z e n e on the_- incorporation of thymidine into DNA.When they f3r studying the effect of benzene on erythro. c-rusame acids radio- piesis using the incorporation of "Fe into autographically I hr after thymidine administra- hemoglobin of maturing red cells as a measure of tion (i.e., the method used by Speck and co- red cell synthesis in the mouse. We found that they also found a decrease in -7after a sin-g-l-e Jose of benzene a reduction in ths the uptake of tritium. Their interpretation, how- Incorporation of "Fe occurred at 1, ever. was that the results may have been due to the t h e when--no inhibition of whlte cell production effect of benzene on synthesis, degradation, re- * ._was a p p e - a r r a t h u u l a a u U c ou n t i n n technloues.- utilization. or pool size of thymidine. However, in Using this technique i t was possible to show, in the event that DNA synthesis was in fact inhibited rcemU t h Steinberg:' -na ' d, early stages in red cell maturation as a result of exposure to benzene, they questioned whether this technique could distinguish involving pronomoblasts and nonnobluts were between direct effects on nucleic acid synthesis more sensitive to benzene toxicity than were stern during S phase and effects of benzene on other cells, reticulocytes, or the process of hemoglobin portions of the cell cycle, i.e., Go, GI, or G 2 . synthesis. Thus. the e f f e a r of benzene on e r y t h m They pointed out that alterations in the cell cycle ma) account for the difference in uptake by Jtenng facton which ngula!e the rate of cell proliferation. Thus. although there may well be decreased nucleic acid synthesis in bone m r n o w of benzene-treated a n n a l s . the damage caused by benzene may a c t u d y haw occurred in any parl of the cell cycle but was observed as an cffea on nucleic acld production within the experimental design BENZENE. LEUKEMIA AND CARCINOGENESIS A a u w and cffea relationship between ben=ne and leukemia has been difficult to establish. Despite some negative reports," it now e r n fairly clear that chronic expolure t o high a n a m trations of benzene may kad to one of wwrd types of leukemia. the most prevalent of which b acute or subacute myeloblastic leukemia.' '*"*" Chronic granulocytic, lymphatic. aleukemic, and crythrolcukemic leukemia and Hodgkin's d h eas~'~-''~''-'' h m dm been reported to rcsuh from chronic bcnune toxidty, but there are fewer of t h e e and in some cam they are not w d l documented. V u i a n i m d Forni" reported that in two studies in France and ltdy involving I total o f 77 f a t a l i t h r u u l t b from chronic benzcne poisonix. approximtely half died from r p b k rnemir urd the remainder from leukemia. Leu- kemia frequently followed aplastic anemia. cells contarning extreme vcsiiulations were obr r v c d . Saita" sugRcstcd that if the marrow is atrophic. leukemic metaplasia may be observed in the liver or spleen. and Millory et a1.16 reported that the sinusoids contained large numbers of undifferentiated cells with many mitotic figures. Although they appeared to be primarily erythrogenic, some myeloid elements and occasionally apparently normal megakaryocytes were observed. Later in the disease they observed the progressive invasion of the pulp with many ivrnature cells. In both marrow and spleen the au'hors noted a general similarity to the picture observed in Hodgkin's disease. The Occurrence of acute leukemia, usually in the later strgt, of benzene tolricity subsequent to r m m w apluia. is characterized by h&h fevers. k m o ~ e s s.erious rapidly developin# anemia, infections, and ulcerations rbout the nose and mouth. Although red a l b and platelets are usually severely &pressed. white all levels may vary considerably from below 5,000 to above S0,000/mm'. As in the case of nonbenzene. induced m t e k u k e m i u , the &ut is usually fat d. mar- ter. Therefore, pa hive erroneously been equated with aphrtic anemia h e n the marrow m@i h m dmron- stntcd that the disorder was in fact rkukemic leukemia, a form of leukemia characterized b y Icucopcnb. The s u ~ t l o n ' ' ~ ' ' that hyperplastic bone marrow rrrociated with benzene toxicity may indicate a preleukemic or leukemic r t r t e suggCR, the necessity of reevaluating pnvlous descriptions of bone marrow in benzene toxicity. For e u m p k . the descdpion by Mdory el d." o f hyperactive bone m n o w In benzene intoxiation c b r t l y p a r d c l s Saitr's" dercription of the bone marrow in u u t e I c u k m l r . Ihe murow displayed extrem proliferation of immature cell forms with n u m r ous mdtlpolrr mitosa. and lrqe multinuclerted &plc who h a w dbplayed r b n o m l c l o n e in the m r r o w h a w not always developed neoplastic disease, while others who did not display chromomal a h n o n n d i t i u were leukemic. Unfortunatety for p u r p o ~of~diagnosis, chromosomal rbnormalitics in t k acute kukemtr are relatively n r e . * Ncntrtklerr, V i d h i and Forni" recommend cytogenic r t u d i a as a useful diagnostic tool in helpin8 to identify benzene toxicity, which may or may not eventually develop into leukemia, provided that exposure to X-rays or other d r u v likely to d t e r chromosomes u ruled out. In view of the apparent role of benzene in Inducia kukemir. an rnalydt of benzene as a a r c h o g e n Lt appropriate. Empirkrl observations June 1975 271 habe demonstrated that aromatic hydrocarbons can be classified according to whether or not they are a r c i n q e n i c . Using this clauification the elec. Ironic structure of these compounds has been studied to determine quantitative parameters uwful fnr predicting carcinqrnicity. The work of ' 'C o u l x ~ ~ 'and Pullman and Pullman,' recently wnfirmed by Hemdon.vO suggests that carcine genicit) of aromatic hydrocarbons can be predicted from cdculations of resonance energies of bonds in the K and L region on the basis of the Itralization theory of chemical reactions. When expressed in terms of chemical reactivity, the theory predicts that compounds in which the K region is chemially reactivt but the L region is not (or is lacking) are carcinogenic. Sims and co-workers' ' - 9 6 have reported that the activity of the K redon resides lar#ely in the ability to form epuudes which can be degraded by revcrll pathways or can react with nucleic acids. Although benzene b thought to be c o n n r t e d to ~1 epoxide." the theoretical calculations predict that it is nonarcinogenic. The prediction of arcinogenicity b u c d on d c u l t t i o n s of resonana energy is s u b c t to error. Despite accurate predictions for such htghly arcinogenk compounds as 3,4-benzo(a) pyrene and others and the a m r a t e prediction of noncu. cinqenicity of 20 b o r n noncarcinogen& it wm shown tha! I6 noncarcinogens were predicted to have a r c i n q e n i c activity while a number of other compounds known to be u r c i n w n i c were not predicted by the theory." The erron may be related lo secondary structural features of the compounds, the route by which they are metabolized, i r n c c u r r y in m u r u r i r q carcine ~ m i c i t yo, r inability t o determine the specific type of cancer in animal modeis. The latter m y be true in the caw of benzene since it has not been possible to reproducibly demonstrate the formation of kukemia in animals after giving ben- me."^" Furthermore, benzene hu not been shown to product s k i n c a n a r or other types of tumors produced by polycyclic aromatic hydrocarbons which were used to develop the theory. The evidencc described by V d i a n i and Fomi" that links benzene to the production of leukemia suggests either that the theory does not predict for induction of kukemia or that benzene is m example of a case where the theory ern. The study of the K region and K r e g h epoxides, hmewr, miy prow to be wcful in predicting how benzene (or its epoxide) may react with nucleic acids either tc depress bone marrow activity or to produce leukemia. In genera! two types of reactions of arcinogens with nucleic acids have been demonstrated."'ii00 Noncovalent interactions include intercalation or external binding. whereas covalent linkages to DNA have been suRgested to occur at guanine moie- While the former may be difficult to detect because of the possibility of removing noncovdlently linked benzene from DNA during isolation and removal of unreacted benzene. the prcsena of covalently linked benzene should present fewer difficulties. Reliminary attempts to defect covalently bound benzene in this laboratory have shown that small quantities of benzene may be bound to liver microsomes. but binding specis ficllly to DNA hu not yet been studied. Detailed studies of fnduction of leukemia by benzene haw been hampered by the lack of a suitable animd model. It hu not been pouible to reproduce the Jingle report of kukemia in benz e n e -t r a t t d mice." Several types of studies might, however, contribute lucfirl infonnation. For example, morphoiogicil d u d i a of benzenetreated animals in which the bone marrow is examined to enluate the Incidence and signific a n e of hyper. vlcnus hypoactive bone marrow in benzene toxicity might provide a model for studying the preleukemic condition in the absence of true leukemia. I t w w l d rlso be of d u e to measun the binding of radiolabeled benzene to proteins and nucleic acids &I bone manow of benzeneintoxicated animals. Thw, biochemical laions krding to k u k e m u in animals may be studied in the a b s e n a of the full-blown disease. IMMUNOLOGICAL ASPECTS OF BENZENE TOXICITY b r l y in this century it was recognized that benzene had an adverse effect on immunological mechanisms. It was demonstrated that suscepti- *'bility to tuberculosis' O Y and pneumonia' O 3 O 4 w a Increased m benzene-treated rabbits. The reports of decreased production of red cell lysins, a&tinms for killed typhoid bacilli and opsonins,"' and the r b w n a of antibactcrhl anti. "*'bodies' O ' in benzene-intoxicated rabbits were JI indications of depression of the production of vuious components of the immune mechanism. Dtvelopnentr in the fkld of immunology have led to studies of the effects of beniene in humans on =vera1 immunological components which have been identified in recent years. Smolik and coworkers' o'l' O' studied a large number of workers exposed to but not seriously intoxicated by benzene. They found that serum complement Icvels. IgG. and IgA were decreased bur that IgM levels did nut drop and were in fact slightly higher. Taken toge!her these observations may explain w h y be n zene-intoxicated individuals readily succumb to mfection and the terminal event in severe benzene toxicity is often an acute overwhelming infection. These authors also evaluated levels of leukocyte agglutinins and found them elevated in selected 'individuals exposed to benzene.' They extend: ed this observation to suggest that in some persons [ne picture of benzene toxicity may in part be acc0unt.d for as an allergic blood dyscrasia. Alterations of immunological function may also play a role in the development of acute leukcmi:. resulting from benzene intoxication described above. Current concepts of immunology suggest that a mechanism referred t o as "immune ''survcitlance"' ' I 1 ' is constantly at work to weed out cells which result from mistakes in cellular genetics or genetic changes caused by carcinogenic agents. Tlie mechanism, while not completely understood, appears to involve a recognition of surface components of abnormal cells followed by immunological destruction of the cell or clone o f cells. Since some forms of benzene intoxication result in hyperplasia of bone marrow with the Occurrence of many bizarre cellular species, it may be presumed that some of these may be neoplastic. D-camsge to immunolo&al mechanisms in benzene toxicitv mav t h m the immune surveillance resoonw. with the i g development of leukemia. BENZENE METABOLISM IN VIVO Although metabolic modifications of foreign chemicals usually result in the formation of more polar, l e u biologically active metabolites, it is not 'uncommon for the products to posses enhanced biological activity.' Thus, prontosil, the first sulfa drug, is metabolically inactive until converted to sulfmiIamide, the active antlbacteria~agent.' " There are also many adverse effects resulting from metabobc activation. Carcinogenesis by 2-acetylrminofluorcne, dimethyl nitrosamine, N-methyl- 4-aminoazobenzene, and aflatoxins. hepatotoxl. city by carbon tetrachloride. and carcinogenesis or mutagenesis by nitrofurans are but a few examples of the activation of foreign chemicals to more toxic compounds.' Is-' I Before undcrtakinp a discussion of the relationship between benzene metabolism and its toxicity. benzene metabolism both in t.vo and in vitro will be reviewed. 'Porteous and Williams' 9 - ' l o a d m i n i w r t d benzene to rabbits orally and using chemical methods determined that the principal metabolites in urine were ethereal sulfate and glucuronide conjugates of phenol, catechol, and quinol. They also detected muconic acid in the urine. More accur3te and quantitative determinations of thc metabolites were performed uhen radioktive labeled benzene became atailable. Parke and ' 'Williams' * I administered 'C-benzene (0.34 to 0.5 ml/kp. orally) to rabbits and recovered 84 to 89% of the dose as radioactivity in the expired air, urine, feces. and body tissues. In the expired air 43% was recovered as unchanged bentene and 1.5% as " C O I . The urine contained 34.5'2. of which phenol accounted for 2 3 . 9 3 , and the remainder consisted of hydroquinone (4.85). catechol ( 2 .2%L hydroxyhydroquinone (0.37). f r o n s -t r a n s m u c o n i c a c i d (1.3%). and Lphenylmercapturic acid (0.5%). The phenolic metabolites were found to be conjugates which were liberated by hydrofysis in strong acid. No free phenols WCTC found in the urine. 'The feces ''and body tissues contained about 5 to 10% of the dose. In other studies Williams and ceworkers' found that 1% of a dose of benzene was excreted in the bile. ''Snyder' studied benzene metabolism as a function of dose in the mouse. When mice were given 'H-benzene at 880 mg/kg subcutaneously in oil. 7m of the dose was recovered in the expired air within 8 hr. The rate o f benzene metabolism was evaluated over a 24- hr period by collecting urine and quantitating the labeled metabolites of benzene from mice given 640 to 8.800 mg/kp of 'H.benzene. Using a double reciprocal plot i t was estimated that a 25-g mouse can metabolize at most approximately 1 mmol of benzene p e r day. The major metabolite of benzene was phenol, but trace quantities of catechol were also identified. fhc phenolic compounds were found primarily as conjugates in the urine, but 8 small pcrcentag (e.g., 5%) of the metabolites was consistently found to be unconjugated phenol. Glucuronide Jum 197s 273 I ~ . ~ , t u n t c tJl i r J b o u t to h!': 111 the div%cand etherfa1 suI1'3te J ~ O U I 2b ?k' Brn/ene mctJholism in the rat &;IS studied by C(1rnlsh JnJ R ! J ~ . ' Gcrardc and Ahlstrum.' :' ' ' -J n ~\'Jl: HIlr.,.+\ Cclrnich Jnd * 'Rb21)' gabe ~ ~ r ~ / L ~I(rI ~ctcrh, cr fed I J Sr3I1~s I~)ch mg,kg Intrjyuritalt1eJ!I\ I .JnJ meajured free phenols. el 11. , deLII tr) I o n 11 I g~ I c. 5 . J n d t o t 31 conjuga t cd phenalls III t h c uriiic In fcd contrulr fiucuronides JL,ic,iinrcd f u r I "T e l f the d t w . nonglucuronide ctmytgJtrq 70". and thr rcmunder was uncunju- gated phenols. In fasted rats the percentage of gju~u:i,n~cferuse tu 487 and the nonglucuronide cunjuptes fell to 44'7. with the remainder free phcnetls Gcrardc and Ahlstrum' used the furmJliun of ethereal sulfare as a measure of ben/enc rnet3bolism. They determined the sulfate ratiu ( i n ~ ~ r g 3 nsiuilfatc,'i)rganic sulfate) in control and treated r3ts. and a decrease in the ratio was u u d as a measure of benzene metabolism. They showed that with dous up to 889 mg,'kp. benzene metabolism was complete within 24 hr. but longer time periods were required for higher doses. Van Rhecs' I f pave rats either 2 or 4 mg of benzene intraperitoneally and measured urinary metabo- lites after 8 and 24 hr by hydrolyzing the conjugates, steam distilling the phenol, and mea- 5,suring it colorimetrically. At thew low doses benzene metabolism was largely complete at 8 hr. Benzene metabolism in d o 5 has not been tud- .re. d in recent years, but inferences from the o der literature can be made. Callow and Hele' '' &e interested in sulfur metabolism a n d attempt- ed to distinguish between ethereal sulfate forma. tion and the production of mercapturic acids. w e given benztn~al-cigg>.se_s.ofl~2~30r mg/kg. As a result. inorganic sulfur in the urine m e a s e d with a corresponding increase in organic sulfur. The socalled "extra" urinary sulfur after giving benzene was reported as either etherell sulfate or "neutral sulfur" (mercapturic acid). Thirty to forty pcrcenf of the dosc was excieted bound to sulc*lr.of which 50 to 75% was ethereal sulfate and the remainder was mercapturic acid. The authors indicated that the ratio of free to fotd phenol in the ,mine suggested !hat 60% of the phenol was excreted unchanged. thereby account- ing for the remainder of the phenol not bound to sulfur. Although glucuronic acids were known at that time. there was n o indication of the finding of these c o m p u n c b in the urine. M-ore recently. ''_--gLBibr-.' iqertensive studies on the metabolism of phentll in the dug. has reported that within 24 hr 5 5 10 80'7 of an injected d w of "C.phenol was r e c o w e d in the Jrinc at doses r a n p n g from 10 tu 100 mp kg. Glucuronides were prumincnt metahditer 31 each dciu Ictel. Fltr eximple. 3 1 discs of 20 lo I00 mg'kp which would be closest to the dusts of benrene reported by Callow avd ''Hele.' (4 1c1 65': of the urinary nieiaholiic~ s e r e recovered 3s glucuronides and I S tu 2 5 q &ere recutered 3s ethereal sulfate. No report of mercapturic acid was made. '' 'Ui I liirnc arid co-workers' *' ' also studied the metabolism of the major metabolites of benzene in. vivo in the rabbit. When they administered I 'C-phenol orally they recovered approximately 9Wc of the dose in the urine almost eqqally divided bet ween phenylsulfaie and phenyl- glucuronide. Approximately 10;: was recovered 3s quinol and less than 1 % as catechol. In contrast to benzene. phenol did not give rise to muconic acid. sugeestinp to the authors that catechol was not a substrate for ring opening. R r k e and Williams' suggested rhar I 2-dihydrobenzcne- 1,2-diol might be a more immediate metabolite of benzene that could give rise to muconic acid. T h e formation of the dihydrodiol has since &en confirmed by Jerina et al.' " Oehme'" administered "C-phenol to d o g . cats, pigs. and goats intravenously and measured the urinary metabolites. The dog excreted approxi- mately equal quantities of free phenol, phenylsul- fate, and phenylglucuronide. In contrast, the cat excreted 80% as phenyl sulfate with the remainder equally distributed between free phenol and the glucuronide. T h e pig converted 60% to the gfucuronide. and 30% was recovered as free . phenol, with very little as the sulfate. Phenylsul- fare reprcvnted 75% of the radioactivity in the gmt urine, 25% was glucuronide, and about 1% was free phenol. When the dose of phenol was increased in these studies. the percentage of total metabolites in each species recovered as phenylsul. fate dccrcaud and the glucuronide portion increas- ed. suwsting that sulfate availability may be limited and that glucuronidation may provide a reserve mechanism following depletion of sulfate. In a more extensive study performed in Williams' laboratory, the metabolic fate of phenol was studied in 19 species including "C-Phenol was given orally at doses of 25 mg/kg except for man, where the dose WPI0.01 mg/kg. and the rhesus monkey, Where 50 mg/kg was . given The majcir route of metabolism in all sprcies was conjrrgatiun, and all species with the exception of the cat and the pig put out both ethereal sulfate and glucuronide conjugates. The pig excreted the tor31 d o g 3s phenylglucuronide aiid the cat as phen!lsulfate (879)and quinol sulfate (13%). Among the other species. some conjupated phenol pnrniril) via the ethereal sulfate route (man, hedgrhog. chicken. jerboa). others favored gI uc u r o n ide for ma t ion (squirrel monkey, capuchin, fruit bat. guinea pig). while in the remunder neither predominated (rhesus monkey, ferret. dog, rabbit, mouse, gerbil, hamster, lemming). Quinol was a significant metabolite in many of the species and exceeded 2Wc of the dose in the ferret, dog. mouse. lemming, squirrel monkey, capuchin, and hamster. Although ihe results of Williams and co-workers and those of Ochme are largely in agreement, some slight discrepancies are apparent which may be related to differences in specific strain or breed of animals, route of administration of the phenol. and dose. ,'Williams and co-workers' 3 o 3 1 also investi- gated the fate of the two minor metabolites of bentcne found in greatest quantity - catechol and quinol. Rabbits were given about 200 m u k g orally of each compound and the urine was found to contain ethereal sulfate and glucuronide conjugates of each. There was n o evidence of the addition of another hydroxyl group to quinol, but trace amounts of hydroxyquinol were recovered after giving catechd. In each case the polyhydroxylated benzene derivatives were excreted as the monoconjugates. The rate of benzene metabolism can be detcrmined by the dose of benzene and by compounds which either stimulate or inhibit benzene ''metabolism. Thus, both Gerorde and Ahlstrom' * and Snyder' demonstrated a dose dependency for the rate of benztne metabolism in the rat and the mouse, respectively. Pretreatment with phenobarbital has been shown t o increase benzene metabolism in the rat by 40%'" and in the mouw by 7 0 % ~ ' 'C~ornish and R y a n t z s reported that SKFSZSA inhibited benzene metabolism in the rat. Toluene inhibits benzene metabolism in both the tat'"v"' and the mouse. &cause benzene is metabolized via the hepatic ''microsomal mixed function oxldar,' it Is not surprising that compounds whfch ttimulate the activity of that enzyme y s t e m might Increase the r3te of bcnienr mefabdism. while thotc u twh react with cytochrome P450 might inhihit lrcnzene metabolism. B E N Z E N E 11ETA BO LIS11 IN I'ITRO Early studies of benzene metabolism in vitro relied on the colorimetric determination of phenol praduction as a measure of benzene hydroxylation. S a k m o t o et al."' attempted to identify the intracellular locus of benzene hydroxylation using differential centrifugation Jf rabbit liver h o m q e n ates. but because they used g forces too low to isolate microsomes they were forced to conclude that the enzyme raided in their 19,OOOg supernatant fraction. They were the first, however, to describe the requirement for reduced pyridine nucleotides in benzene hydroxylation and reported that phenol was the principal metabolite. They also showed that the enzyme was inhibited by heavy metals and was sensitive to dialysis. Although these key observations were reported relatively early in the study of benzene metaboI t m in vitro. they were not appreciated because they were published in Japanese in a rather obscure and inaccessible journal. Using a similar ''procedure, Posner et al.' demonstrated that bcnsene was converted to phenol in microsomes isolated from rabbit or dog liver. Because of the lack of sensitivity and the possibility of interference by other phenolic com. pounds, the colorimetric method of phenol determination was not considered adequate for detailed studies of benzene metabolism. Thus, Snyder et d.I3' instituted the UK of radioactive labeled benzene to measure benzene metabolism in subcellular fractions of liver from rabbits, rats. and mice. Both hydroxylation of benzene to phenol and subsequent conjugation to form either phenylsulfate or phenylglucuronide were measured in elther whole hornogenates or 9.OOOg supernates, whereas only phenol fornation occurred in isolated microsomes. In 9,WOg supernates about SO96 of the phenol was conjugated but upon addition of ATP and sulfate o n r 90% of the phenol w a converted to phenylsulfrte. In conlrut UDPC @~curonicacid or its precunon ATP, UTP, NAD,m d glucose were less effective in promoting the formttion of phenylglucuronide. f h e rate of benzene metabolism in these studies rppcared to be related t o the d e g m of conjug, , lune 1975 27s . tiun heLausc wtwn ATP and sulfate were added m r e benzene was metabolizrd. 140.1 4 I It was suggested that p r h a p there was either inhibition of benzene hydroxylation by competition of phenol with bcnzcne for 8 Ste on benzene hYJrt1x)lase or phenol exerted a negative f e d b a c k on the reaction. Under these circumstances conju- gatittn would remQve phenol and thereby stimulate the reaction To test this idea sodium fluonde was added io these peparations. Sodium fluoride inhibits ATPase and would therefore amplify the effectiveness of small amounts of ATP. I t dso has the effect of inhibiting ethereal sulfate forma- ~ I U ~ I . " T~he result was that fluoride prevented the conversion of a large percentage of phenol to phenylsulfate but the increase in benzene metabo. lism was not altered. The stimulition of met8bo- lism appeared to be a function of the effects of ATP and fluoride on benzene hydroxylase rather than on conjugation. and. indeed, when added to isolated microsomes both produced 8n increuc in the rate of benzene hydroxylation. The mechtn- ism has yet to be determined. but the effect is not observed in the munt of the metabolism of chlorpromazine, zoxazoladne, neoprontosd, p nitrobenzoic acid, aniline, or N-methylmiline.' I I t was of interat that the oxidation of aldrin to dieldrin was stimulated by ATP and F in vitro. Liver microsomes contain 1 mlrtiwly nonspeci- fic enzyme system a i l e d the mixed function oxidase which is primarily responsible for the metabolism of a great m y xenobiotic a m - The system requires d e c u l a r o x y g n and NADPH for activity. Mixed function oxidase activity revolves about a = r i a of reactions of a heme-containing protein c d e d cytochrome P450. which is the active rite at which both ~ubstratc"~" 47 and oxygen The substrate reacts with oxidized (i.e., Fe'") cyto- chrome P450. and the resulting enzyme-substrate complex Ir reduced to the Fe* form by reducing equivalents originating from NADPH and tnns- mitted via a flavoprotein enzyme called NADPH- cytochrome P4SO-reductuc.' so.isi Atema- tively, lome of the electrons may come from a second source of reducing cquivrlcntr involving NADH, another flrvoprotein &led NADWcytG chrome bS-reductase, and another hemeprotein .'d l e d cytochrome b, "*I" The latter b itself incapable of reacting with oxygen, b i n d h g sub- strates, or a t d y r l n g hydroxylation, but It m y rid in the reduction of cytochrome P450. The sequence uf events continues with the reaction between reduced enzyme substrate complex and oxygen; the substrate undergoa hydroxylation and the cytochrome is reoxidized. The demonst ration of benzene metabolism in liver microsomes strongly suggested that the mixed functian oxidase w u benzene hydroxylase.' 5 7 Several p i e m of evidence taken together indi- cate that benzene is hydroxylated by cytochrome P450 and the mixed function oxidax. Benzene hydroxylation occurs in liver microsomes of rab- bits, rats. m d mice and requires oxygen and NADPH.1'6*"8.'3P Benzene reacts with cytochrome P450 to yield a Type I spectral change indicative of the formation nf an enzyme-substrate complex and with cytochrome P448 to yield a Type RI spectral change in much the same manner u many other substrata for the mixed function oidaJc.l 3 6 5 8 Aniline, metyrapone, aminopyrine, and SKFSZSA, 111 of which inhibit the metabolism of other compounds by cytochrome P450 or interact with cytochrome P450, inhibit benune metabolism.' " Cytochrome c, which inhibits mixed function oxidase reactions appar- ently by diverting electrons from cytochrome P450,'" du, inhibits benzene hydroxylation.' 36 Benzene hydroxylase, like other microsomal hydroxylases. is inducible, and its induction will be described bdow. Finally. benzene metabolism b inhibited by carbon monoxide, and the inhibition is best reversed by light at r wavelength of 450 nm.'36 From these data it seems fair to conclude that benzene hydroxylase is a form of the mixed function oxidase. The mechanism of benzene hydroxylation by the mixed function oxidase has not yet been determined largely because of the difficulties encountered when trying to isolate and purify the membrane-bound components of the microsomal enzyme system. Studies by Jerina and Daly' and their associates have concentrated on the chemical aspects of the problem and have suggested that the reactions probably occur via the formation of an u e n e oxide intermediate. Upon the addition of benzene oxide to a ndcrosomd preparation, the products of benzene metabolism were formed.' " Benzene oxide production hu not been demonstrated in microtoma probably becawe of its extreme lability, but nrphthdene oxide WIJ recovered when mphthalene, 8n aromatic hydrocarbon cl0r;ely related to benzene, WIJ incubated with I i n r minolrorncs.''O On theoreticd (pounds, HamJton'" has proposed that it would be expected that mixed function oxidation reactions proceed through arene oxida. Furthermore, u e n e '-' ' ''o x i d a of a number of aromatic hydrocarbons haw been reported .9 * I Therefore, benzene oxide appcan to be a likely intermediate in benzene metabolism. The products of arene oxide degirdation resuit from either enzymatic or nonenzymatic re%- tions.' ' Noncnzymaticdly benzene oxide can undergo isomerization to form phenol by either of two mechanisms, one of which b favored at low pH while the other h pH independent. Since both in vivo and in vitro phenol ir the major metabolite of benzrne and biological systems function in the n n e of neutrdity, the latter appears to be the preferred mechanism. Enzymaticdy benzene oxide may be hydrated to 8 dihydrodiol by the action of epoxide h y d ~ e a ' 4 D ' ' 4 ? a ' s foUowad by subsequent reductbn to catechol. The slgnifi- a c e of the latter p t h w a y hu yet to be determined since very little catechol fr formed in the course of benzene metabolism. Another en zymatic raction ir thc transfer of glutathione to arene o x i d a by the enzyme arene oxide- glutathione transferase. Pehrp the mort r b i f i - cant p t h w a y for the degradation of benzene oxide b the reaction with c d l u h r nucleaphila such as nucleic acids or protdna which could result in cellular dunage indicative of benzene toxicity. Thus, the metsbolic fate of benzene CUI be Vifflllized as the fonnltion of i n rrene oxide foUowed by the r e a m n p m e n t t o the less chemi- d y reactive phenol, the interaction of the oxide with cdlular nucleophiles, or the enzymatk con- version to either the dihydrodiol or a pemercap turic add. The hepatic minotomrl mixed functlon oxi- dw tyrtem can be stimulated b y enzyme induc- tion by at Iuat two *nerd dlrser o f compounds: ( I ) pdycyclic r m a t i c hydrocarbons m d (2) phenobarbital plus 1 number of other compounds which act aimiIady."' The former appear t o induce the formation of a type of heme protein a l l e d cytochrome P448, w h e r e u phenobarbltd '' ''i n d u c e s t h t f o r m a t i o n o f c y t o c h m m e P4SO.I Benzene hydroxylase r t l v f t y in vitro increases lner t r t a t i q rnirmlr with pheno- b u b l t d or I m e t h y l c h d m t h r c n e , i polycyclic aromatic AdmMt n- don of benzene Iklf o r DMSO rL0 l n c r r u t t the n t e of benzene '*,*rnetibo~igmI.~**' Whcn benzene or DMSO is administered subcutaneously or intraperitoneally, benzene metabolism in liver preparations increases but there is no increase in 'c y t o c h r o m e P4 SO levels.' , I * I Recently Norpoth et ai-"' reported that exposure of rats to benzene npor at about 450 ppm for IO days resulted in an increase in cytochrome P450 levels of about 65%. and Drew et al.'"' reported t h a t exposing rats to levels of benzene in the atmos- phere above 4,000 ppm for 4 hr/day fur 3 days resulted in an increase in microsomal benzene metabolism. Newtheless. the reports of increases in b e n u n e metabolism by benzene or DMSO in the absence of increased levels of cytochrome P450 suggest that other facton may play a role in determining the rate of benzene metabolism. When benzene U added to liver microsomes, it b pasible t o observe a Type 1 spectnl change typical of substrates for the mixed function oxidase.' " When the technique of Schenkmm et d"' for titrating microsomes t o obtain the apparent s p e c t d dissociation constant (K,) was applied, it was found that In liver microsomes from benrrne-treated mice there was no change in the IC, but the murimurn change in o p t i d density (A OD,,,) v u increased. By the m e token kinetic r t u d i a showed that metabolism in liver microsoma of benzene-trated mice was chuac- terized by no change in the KM for benzme but the V, increased by about the same percentage (a16046of controls) u the A OD,,,. In addition to increasing the rate of benzene metaboUsm, parenteral administration of benzene dzo increased the rate of zoxazolamine hydroxylation urd the reduction of p-nitrobcnzoic acid in rats."' Stimulation of the metabolism of both benzene' " and aminopyrine' '3 has been reported followin8 exposure of rats to benzene in the atmosphere. Therefore, benzene appears to function as a microsomal stimulant which can increase the rate of drug metabolism without necessarily increasing cytochrome P450 and as a result p o x s questions regarding the nature of the induction which may be dealt with in the context of our understanding of microsomal enzyme induction. In the course of developing current concepts of hepatic microsomal enzyme induction, it became clear that many drugs, among which phenobarbital md the polycyclic aromatic hydrocarbons are mort prominent, were capable of initiating protein g y n t h e b in liver. Increased microsomal pro- h c n a r d hcorpontion of amino rddr intu hepatic microsomal protein.I I J - I l 7 .increased j c t ~ n t yand synthesis of nucleic aclds and enzymes that mediate their prevention of induction by inhibitors of protein synthesis.' "* ' " * I " proliferation of smooth endoplasmic "-'reticulum ISER).' " and increased synthesis of cytochromes P450 and P448'510a00~'h0a've bccn determined during enzyme induction. Induc- tion by benzene is characterized by an increase in metabolism and in incorporation of amino acids iato m i c r o s o d protein within 24 hr of a singk "*'d e . but no prdiferation of SER is observed a t h a t time.' " Af ter I or 2 weeks of benztne treatment, SER appcarr to k proliferated and the mtabolisms of benzene, zoxuolrmine, and neoprontod remain slightly stimulated. whereu those of hexobarbital and p-nitrobenzoic acid are d e p r c w d . ' I` Bentcne induction is characterized by requiring a longer time m d produchB leu proliferation than phenobarbital. In contrast, it is more similar to polycyc1ic aromatic hydrocarbons, which also i n d u a rapidly and d o not ow proliferation of SER at the time of induction.' " There may be a greater similarity to dieldrin, which after several weeks c a u w the formrtion of hypertrophic, hypoactive smooth endoplasmic In i l l , thex obrcnrtions suggest that there does not wem to be a relationship between the degree of proliferation of SER and the rate of benzene metrboiisn. Since hepatic microsomal enzyme induction involves protein synthesis, the data w s t that a t leut two different types of protein m y k yntherizcd t o account for incream in the rate o f '`benzene metabo1iun. The furding by Gonuun et d.' that the increase in k n u n e metabolism after induction with benzene vu not a c c o m p k d by e k n t e d levels of cytochrome P4ul is consist- ent with induced synthesis of a minor component of the m i c r o r o d cytochrome P450 population which is responsible for benzene hydroxylation but which represeenu a relatively md fraction of the total m i c r o s o d cubon monoxibe binding heme proteinr Thus,'a sizzable percentage increuc in that component might p undetected when measuring cytochrome P450 but become apparent only when measuring binding s p c t r r . Recently, Norpoth et ai.' " demonstrated that after exporc in8 rats to b m z m c vapor for 10 days (5 h l b y ) , cytochrome PI50 knls in liver lncrcucd 65%. Therefore, benzene can increrw cytochrome P450 kvcb but not r8pid)v enough to obrcm the effect within 24 h r , the time at which benzene can increase the rate of its metabolism. An alternative posibiiity was suggested by R ~ m r n e r ' ~ 'several years ago but has not yet been evaluated. He showed that phenobarbital stimulated the metabolism of hexobarbital and amino- pyrine and DDT stimulated the metabolism of hexobarbital, bijt in neither case was there an increase in cytochrome P450 levels commensurate with the increav in the rate of metabolism. He also obscwed that male rats metabolized hex+ barbital faster than female rats but that the differena could mt be explained on the basis of cytochrome P4SO levels. In each case the rate of metabolism was more clokly associated with the optic4 density changes in the binding spectra than with cytochrome P450content of the microsomes. Increases in binding spectra following enzyme induction have dm been reported by other workers.' "J ~" R`emm~er'o'~ suggested that reprdless of the effect of inducing agents on cytochrome P450 concentrations, the relationship between the rate of metabolism and the binding spectmm may be - u r d by the induced synthesis of a r o c d e d "binding protein" which facilitates the binding of the substrate to the cytochrome. If the nte-limiting step in the hydroxylation of benzene is the rate at which the enzyme substrate complex CUI be reduced,206 then a protein which increases binding may hasten the rate of reduction and thereby stimuiate bentcne merabolim. Both of these possibilities d a m further study. RELATIONSHIP BETWEEN BENZENE METABOLISM AND TOXICITY Relatively few studies have been aimed a t correlating benzlne metabolism with k n t e n e ' *'toxicity. Following the definitive studies of F'arke and Willhms,l in which the metabolites of benzene were identified as phenol and polyhy- droxybted phenols, Dustin"' suggested that the metabolites might be responsible for benzene to&ity throufi either of two mechanisms. In one, quinme-yielding metabolites such as cate- chol, quinol. or pyrogUol could react directly with chromosomes and interfere wtth mitosis. An dtemrtive p r o p a d was the following sequence of events: (I) depkdon of d f a t e , derived from sulfur amino acids. for p u r v of conjugation, k a b g t o (2) ntbwgumt dcpletim of d u t r t h h e In bone marrow, resulthg in (3) disturbances in redox rerctrons in bone marrow, leading to (4) bone murow depression. No evidence has yet been developed to support either of these suggestions, bur they represent some of the first thou@t indicating that benzene metabolism may play 8 ' 'role in benzlne toxicity. Nomiyama et II.'"-' reported that young rats dllrplayed a high rate of benzene metabolism in vitro and were more susceptible to benzrne given subcutaneously than old rats in which the rate of benzene metabolism was slower. They demonstrated that 3-amino-l .Z,Ctriazolc, which d u b i t e d benzcne metabolism in rat liver h o m e genates, protected against benzene toxicity. They therefon concluded that a metabolic product of benzene was responsible for benzene toxicity rnd proceeded to test wverrl benzene metabolites for their potential as hemotoxic rgents. Among phenul, catechol, quinol, hydroxyquinol. rmm- transmuconic rcid, D .L-phenyhercapturic rcid, and potassium phenylsulfite, only c t t e c h d appeared to depreu bone marrow function by causing anemb rnd leucopenia. At about the same time Ikcda" exposed rata to benzene in the rtmosphere rt 1,ooO ppm for 7 hr/dry, 5 drydweek and showed that the order of increasing sensitivity to k n z e n e based on depres- sion df white ccu counts was adult mdes > y w n g males > rdult femdes > young fermles. He then evaluated the levelr of several enzymes invdved in benrrne metabolism. Aryl4hydroxylasc was assumed to be the enzyme that h y d ~ o x y h t e s bmzme, and ib activity was determined using aniline u the substrate. Ethereal sulfate and ducuronide-forming enzymes were e v d w t c d udng pnitrophenol 8s the substrate. He Loncludcd that the best correlation between bcntene toxicity and metabolism could k made between the rate of ethereal sulfate formation and toxicity. Thus, the relationship among the various groups of rats with respect to decreasing ethereal sulfate f o m u t i o n was Identical to the relationship stated rbon for increasing sureptibility to benzene. No conelr- tion between metrholism m d toxkity for the other enzymes was found. Ikeda s u w s t e d that Nomiyuna's results may have differed from his o m becaurc N o m i y a m u r d w i ~ d eh m q n r t e s rather thrn the cell frrctims fn which q e a t a t enzyme activity c d d k o b r n e d and kuurc Nomiyanr f d e d to fortlfy h& h o m g n r t n with pyridine nucleotides. It b rppuent, homer, that Ikeda, in iftempting to study enzymes involved in bcnztne metabolism in vitro. used neither benzene nor its metabolites as substrates. Although the mixed function oxidase is the enzyme that hydroxylales both benzene and aniline, there is no reason to a s u m e the rates of these reactions will be equal nor can it be assumed that the rates will necessarily vary among the groups of rats that he studied in the same way for both substrates. T h e rclationship of these studies to events in humans is even more tenuous when it is considered that many instances of sex diflerences in drug metabolism have been reported in rats which do not occur in other species.'" Although it was previously suspected that women were more susceptible to benzene toxicity than men, that idea has not stood the test of time rnd further experience.' l k e d a " s * 2 ' s then went on to study benzene metrbolism in vivo and reported that rats treated with phenobarbital metaboliixd more benzene and were more resistant to benzene-induced leuco- 'penia. These results were later confirmed by Drew et ai.' " Ikeda' therefore suggested that metrbolism of benzene was a detoxifying procedure and that inhibition of benzene metabolism should result in increawd toxicity. I t must be argued that In fact these studies could not distinguish between toxicity by benrrne or 8 metabolic product because the increase in metabolism produced by phenobarbital might either have detoxified benzene or hastened the removal of a toxic intermediate, a mechanism similar to that o d d n d l y suggested by Ikeda.' " In this hborrtory we have studied the relationchip between benzene m e t r b o l i a and toxicity by rdmmbterhg labeled benzene to mice and (1) usins the Irbded metabolites in the urine as a masum of the rate of benzene metabolism and (2) using the reduction of the incorporation of -<'Fe given the next day into erythrocytes of the same mice as 8 measure of hematopoietic toxicity produced by that dose of benzene.' " When the mice w e n given toluene, 8 competitive inhibitor of benzene metsbofism. the rate of benzrne metabolism was reduced and "Fe uptake into erythrocytes increased, indicating that benzene toxicity was dlevirted. In dmdrr studies in which phenobarbital WRY @veri to mice to increroe the activity of the mixed function oxidase, mice with higher ntcs of benzene metrbolism demonstrrted I w a t e r decrerw in "Fe uptake than mice in a h less bentcne wu m e t r b o h d . These studies .. Jon 197s 279 would rend to support the view expressed by Nomtyama that a metabolic step was important In causing btn7cne toxicity. A common thread seems to run through the studies renewed above. In each case the effect of benzene on a parameter of bone marrow activity was conelated wth the rate of benzene metabolism either in vivo or in vitro using liver prepara- tions Some recent studies sugvst that these approaches might be modified to yield further ' `-''information Acetarmnoph`n' I induced liver necrosis appears to be directly related not only to the rate of acetaminophen metabolism but also to the extent of acetaminophen binding to hepatic protein. Stimulation of acetaminophen metabolism by phenobarbital increases both necrosis and binding; inhibition of acetaminophen metabolism by piperonyl butoxide or cobaltous chloride ''protected against necrosis while &creasing bind- ing. Brornobenrrne' dso produced hepatic necrosis, but inhibition of bromobenzcne metabolism by SKFSZSA coincided with reduction of liver damage. The effect of enzyme induction on bromobcnzrne-induced liver hepatotoxicity, however. depended on the metabolic pathway induced. Thus, phenobarbital stimulated the formation of a hepatotoxic metabolite, while 3-methyl c h d r n - threne h a e a s e d the activity of a detoxifying pathway. The rate of benzene m e t a b o h m may aflect bone manow &pression by a mechanism similar t o that by which acetaminophen or bromobcnztne produces liver dun-. Furthermore, there may well be both &toxitying pthways as well as pathways which lead to bone marrow depression which could help to explain differential sensitivity of humans t o b e n u n e as a result of enzyme induction. In any event, the role of c o n l e n t binding to subcellular constituents in benzene toxicity should be evaluated. Although there u a m i d e r a b k suspicion that benzene mctabolisn plays I role in benzene toxicity, there is p o d reason why the evidence is not yet definitive. The study of bcntcne mctaboLimn whether in vivo or in vitro hau In actuality been a study of events that occur m the liver. The liver h responsible for most of the mctabolisn of benzene, m d the mttrbolites of benzene in the urine are essentially the result of hydroxylation and conjuytion in liver. However, benzene toxi- city is manifested in the bone mrrow n t h c r thm the liver. It must either be ufued that a toric mctabotfte travels from the Uvcr t o the bone marrow or that the metabolite is indeed formed in the bone marrow. If benzene oxide is the active metabolite, its chemical reactivity is too great for it to survive transport through the circulation. Phenolic metabolites might weU be transported free or as conjugates which may be hydrolyzable in the bone marrow. However, there is some ' ' 'evidence that conjupates of benzene are neither degraded nor are they toxic.' I-* It appea IS, therefore, that although the liver m a y serve as a good model for the study of the enzyme systems which metabolize benzene, the most profitable tissue for rtudyiqj the role of benzene metabolism in toxicity is the bone marrow. The techniques are difficult, and bone marrow h not available in large quantities. It may be necessary to use the methods of tiah culture. Amom the problems to s t d y are the mctaholim of benzene in bone marraw and the effects of the mtabolites of benzene formed either in the liver 01 the rmrrow on bone marrow activity. In any event, a point has been reached in the study of benzene toxicity where we must recognize that there can be no more direct approach to studying the disease process than to investigate it in the o r y n where it occurs. SUMMARY AND CONCLUSIONS Although the morphological aspects of damage to hematopoietic organs caused by chronic ben- zene exposure have been understood for mmc time, the mechanism by which benzene acts is not known. The evidence suggests that benzene inhibits the maturation of early blood cell precursors; Le., the Arneth count is shifted t o the left in the leucocyte writs and maturation of pronormoblasts and normoblasts is inhibited in the erythrocyte wries. In advanced stages the result can be pancytopenia due to bone marrow aplasia. DNA syntt-rsis is reduced in bone marrow of benzenetreated animals either because of inhibition of enzymes involved in DNA synthesis or because a leion revealed as reduced incorporation of triti- ated thymidine in DNA occurs at some point in the a l l cycle. How benzene mediates this inhibi- t b n is not yet ckar. AI t h o u g h e p i d e n i o l o ~ c d studies among worken in industries whert benzene exposure is a mud hrn failed t o demonstrate a correlation between the Incidence of kukemia and benzene exposun, many individual CUCI of leukemia have k n linked to benzene. The suggesttion that . . hyperplastic bone marrow sometimes seen in benzene toxicity is indicative of the preleukemic state and the concept that aleukemic leukemia may he more frequent in benzene toxicity than was previously recognued are ideas that must be more fully explored. The evaluation of chromosome abnormalities in benzene-intoxhled individuals might be useful in determining leukemia, but I t must be recalled that acute leukemia, which is frequently related to benzene exposure, yields c h r o m o r o m abnormalities relatively rarely. Fur- thermore, abnormal chromosomes are not necessarily prognostic for leukemia. Benzene has been known to repress immunoIqical mechanisms for many years. Recently it was demonstrated that specific immunoglobulins such as IgA and IgC, as well as serum complement are depressed while fgM is elevated in benzene intoxication. Two obnrvatims in benzeneintoxicated individuals may result from h u n o . logicd damage. Benzene-intoxicated individuals often suffer from serious infections which they are unable to combat and thus may be terminal. Furthermore, in some instances of benzene toxi. city the "immune surveillance" mechanism which appears to be partly responsible for preventing the growth and development of neoplastic tissues may not function and the result may be benzeneinduced leukemia. Benzene is hydroxylated hy the microsomal mixed function oxidase to phenol and other hydroxylated benzene derivatives probably via the intermediate formation of an epoxide. The epoxide may either rearrange to yield phenol. conjugate with flutathione to yield a premercapturic acid, be hydrated to epoxide hydrase and reduced to catechol, or it may react with various cellular constituents and thereby produce its toxic effects. Despite some contrary evidence. there are considerable data to support the concept that a metabolic step yielding a toxic intermediate is necessary to produce benzene toxicity. Thus, inhibition of benzene me tabohan protects against benzene toxicity. Since the bulk of these studies has been performed using whole animals or liver preparations, it is essential that further studies revolve around the metabolism and toxic effects of benzene in bone marrow. ACKNOWLEDGMENTS The authors wish to thank their colleagues Drs. E.W. Lee and C.M.Witmer for discussions during the preparation of this review. This work was in part supported by a grant from the USPHS. National Institutes of Environmental Health Sciences CESOO322 ). REFERENCES I . Hunt-, D., The Disapres of Ocrrrprrrionr, Little, Brown & Co.. Boston, 1962. chap. 1- 4. 2. Rmutini, B., Discores of k'orkm (De Morbis Artiflmm). revised. trrnshted. and annotated by Wrbht. U'. C-. Hafner. New York. 1964. 3. Hunlton, A,The $rowing menace of benrene (benzol) poisonirrg in American industry, JAMA, 7 t 627. 1922. 4. Santarnn, C C., h r chronischc Veqiltung mit Steinkohlenthecrbtnrin: v u Todcsfalle. Aroh. Hyg. Bin!. 31. 336. 1897. 5. Selling. 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