Document ByJgNGOOkqNX15y3xEO4VKGLm

\. + -. S. P. Tuckcr et al. b< lphide by thc rat. Effect of Ic. Chcm Diol Interact t microsomal drug-mmccedings of the European :, Amsterdam. American Press Ltd.. London and r :patoxicity in rats cxposcd :ffect of dict and induccrs ates and on metabolism of .oro-2.2-bis-f f-chlorophcn sensitivity of rats to -hide used in combination or sheep. Res Vet Sci he Iowa State University cory animals. Vct Rcc 0 N 65 Arch. Toxicol. 45. 297-305 (1980) Effects of the Principal Hydroxy-metabolites of Benzene on Microtubule Polymerization Richard D. Irons and Douglas A. Neptun ! Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina, NC 27709, USA I I Abstract. The principal hydroxy-metabolites of benzene - phenol, catechol i -and hydroquinone possess characteristics and produce toxicity similar to I f those reported for certain inhibitors of microtubule polymerization. In this study we examined the effects of phenol, catechol and hydroquinone on purified microtubule polymerization and the decay of tubulin-colchicine I binding activity. Hydroquinone. but not catechol or phenol, inhibited i microtubule polymerization and accelerated the decay of tubnlin-colchicine ! binding activity. The latter effect was shown to be dependent on the 1 concentration of GTP.Hydroquinone did not directly complex with GTP or i ATP but bound to the high molecular weight fraction of tubulin. I ! Concentration ratios of hydroquinone to tubulin resulting in altered activity were low, 'suggesting a specific interaction, presumably at the tubulin-GTP binding site. The acceleration of tubulin-colchicine binding activity decay was completely prevented under anaerobic conditions, indicative of an ! oxidative mechanism. These studies suggest that hydroquinone, which auto-oxidizes, may interfere with microtubule function, nucleotide binding or both and that this mechanism may be involved in eliciting :he wide range of cytoskeletal-related abnormalities observed in cells exposed to benzene in ! vivo or its metabolites in vitro. -Key words: Benzene - Hydroquinone - Microtubules GTP - Auto-oxidation. Ii I Introduction Microtubules, which are polymers of the tubulin molecule, are known to participate in several structurally related processes within eukaryotic cells. Microtubule integrity is a requirement forspindle formation during cell division, maintenance of cell shapc, intracellulur organellc movement and secretion of cellular products (Bryan 1974; Lacey ct ai. 1968: Katz, 1972). Microtubules, I 0340-5761/ti0/0045/O~97/S01.80 I I i I i I ! ! i I I ! I i ! --I f I i I 298 R. D. Irons iind D. A. Neptun microfilaments and their associated proteins are also involved in the regulation of cell surface receptor mobility and cell growth (Edelmann 1976; Mc-Chin and Edelman 1980). A number of different compounds are known to inhibit tubulin polymerization or to accelerate tubulin depolymerization including: colchicine. vinblastine. diphenylhydantoin and selected sulfhydryl reagents (Wilson et ai. 1973: MacKinney et al. 1978; Himes and Himes 1980: Mellon and Rebhun 1976). Exposure of cells to these agents in vivo or in culture results in metaphase arrest. uneven nuclear division and chromosomal abnormalities (MacKinney et al. 1978; Himes and Hirnes 1980; Deysson 1975). Additional compounds which block division. bind to tubulin or inhibit tubulin polymerization possess structures similar to catechol or hydroquinone, the principal dihydroxymetabolites of benzene (Traganos et al. 1980; Misurni et al. 1979). Administration of benzene to rats or rabbits results in cycle specific changes in the kinetics of proliferating cells in bone marrow indicative of G2/Mphase arrest (Irons et al. 1979; Irons, 1980; Muirhead, et ai. 1980),and a number of studies have reported abnormalities of cell division consistent with mitotic arrest following benzene . - exposure (16). Phenol, catechol and hydroquinone have been identified as mitotic inhibitors of cells in culture as well (Parmentier and Dustin 1953). It is generally accepted that benzene is not directly toxic,but that toxicity is dependent on the metabolism of the compound to a reactive species (Laskin and Goldstein 1977). Recent studies in our laboratory indicate that polyhydroxy-me- -tabolites of benzene hydroquinone. catechol. benzoquinone and 1.2.4-ben-zenetriol suppress mitogen stimulated lymphocyte response in culture at non-cytotoxic concentrations (Wierda et al. 198(i), and under physiologic conditions both hydroquinonc and 1.2.4-bentenctriol are subject to auto-ox- idation (Greenlce and Bus 1980). In this paper we report the inhibition of microtubulin polymerization and the accelerated dccay of tubulin-colchicine binding activity (TCBA) by hydroquinone in vitro. Phenol and catechol were also investigated but had no effect on these processes. Hydroquinone does not alter the affinity of colchicine bindins to tubulin but apparently competes for sulfhydryl dependent GTP-binding sitcs on the protein. This is prevented under anaerobic conditions, indicative of an oxidative process. Similar effectson GTP binding to tubulin have been reportcd for selected sulfhydryl reagents which preferentially bind to -SH groups at the GTP-binding site at higher rates than to other -SH groups on thc proteiii molecule (Mann et al. 1978). These findings offer a possible explanation for alterations in cell growth or differentiation resulting from exposurc to benzcnc or its metabolites through disruption of rnicrotubulcs. interference with GTP other nuclrotidc depcndcnt processes or both. hIaterials and F)Iethods Cltemicols. ("C]h~droquinone. [ "Clcatcchol and [ "C)phenol (37.9 Ci mol) \curt' oh1ained f r r w Midwest Research Institute clnd [ 't llcolchicins (23.2. Ci.mmol) Irom S e w EnS1;lnc.i Nuclear. Colchicint and catechol uzru 0ht;lincd Irom Sigma and hydroqoiiionc and phcniil from AIJrich Efftots of Bern !solution oj (2250g)asdcs anesulfonic a& GTPJ.The pH Measurement oj measured turk spsctrophotorm corrected for Im merhod (Gom expcrimcnts. A S S Uo~f Colt%. assaycd by gd i addition of GTP 3.7 X 1O"'beEqL binding was as Eleclrophores& out using the amino-mcr h a m paper chromam and Isherwood Results Hydroquino pol ymerizari. Conccntriitio. (IC+,,)\vas 1 iCColchicine it affinity ofin: - 01 < 02 j,,p-'-c I" I. Fig. 1. Ilflcct ti! tlrciquinoiiz I httcr. nuinta S;iiiiplc* \\ere ;il1w*rlwiicc mt. r\\ c' c L I ir r LTWL * ind D. A. S'cptuii 8 ! the regulation ;Mc-Chin and bulin polymerig: colchicine. (Wilson et al. 7. and Rcbhun i> in metaphase {MacKinney et -1pounds which zation possess aydroxymetabministration of the kinetics of s t (Irons et al. j have reported ming benzene il identified as Dustin 1953). that toxicity is ies (Laskin and llyhydroxy-meand 1.2.4-bcli: in culture at cr physiologic :ct to auto-ox- ization and the ) by hydroquiid no effect on :hicine binding P-binding sites dicative of an been reportcd I groups at thc In :he protein vplanation for .re to benzene nce with GTP Effects of Benzcnc iMctaholitcs on hlicro!ubulc Polymerization ,299 Isolurron flf Rur Drain Mirrarrcbrrlin. Rat brain tubulin was isolatsd from m s k Fischer-344 rats ( 2250 g) as dcscribcd (Borisy et al. 1974, 1975) using PEG buffer (0.1 XI piperazine-N.N'-bis(2-ethanesulfonic acid): 1 mM EGTA (ethylene glycol bis(~-aminocthglethrr)tetra~cctIa~cid: 2.5 mM GTPJ. The pH of all incubations was monitored and maintained at 6.93 2 0.02. Mea.turemenr of Tubuliri Polymerizariori. Thc temperature dcpendcnt polymerization of tubulin was . measured turbidometrically (Gaskin et al. 1975) at 350 nm using a Gilford Stasar model I11 spectrophotometer equipped with a temperature controlled flow cell. All mcasurcments werc c corrected for buffer and reagent absorbance. Protein concentrations werc dctermincd by the biuret method (Gornall et al. 1949) and adjusted with buifer to 1.7 mg/ml for polymerization experiments. Asmy of Colchicine-Tubulin Bindirrq Deccy. Colchicine-tubulin binding and TCBX decay were a s s a y d by gel filtration as described (Wilson et ai. 1974) using the isolation buffcr without the addition of GTP (PIPES). For determination of colcliiciiie binding. 0.1 pCi of [?Hlcolchicine (1 Ci = 3.7 x 10'" becquerals) was added in varying concentrations to the incubations. [L'C]hydroquinone binding was assayed using the same method (0.1 pCi,'assay). Elecrruplioresir. Sodium dodscyl sulfate (SDS)polyacrylamidc disc gel electrophoresis was carried out using the method of Shapiro et al. (1967) modified using TRIS [tris(hydroxymethyl) amino-methancll buffer. 0.4 M.pH 8.8. "C-labelled metabolite-nucleotide binding was assayed by paper chromaiography as dcscribcd (XlacKinncv et al. 1978) and nucleotidcs identified using Hanes and Ikherwod Ycagent (Zweig et ai. 1972). Results Hydroquinone, but not c;itcchol o r phcnol. inhihited purified microtubuiin polymerization in vitro in a concentration dependent manner (Fig. 1); the concentration of hydroquinone producing a 50% inhibiton of polymerization (ICs,,) was approximately 10-fold greater than that of colchicine (Fig. 2). Colchicine is thought to inhibit polymerization by binding to or altering the affinity of interaction sites between tubulin molecules (Wilson and Meza 1973). c obtained from :nylund Suclcar. I I f~rom Xldrich. MIN AT 37'C Fig. 1. Effect of hydroquinone on the pol!merizJtion o f microtubulcs. Vnriablc amounts of hydroquinone (0. 1. 10. or 15 pl) werc added to 1.7 mg of purified rat brain tubulin in 1 ml of PEG buffer. maintained ai 0" C. to give final concentrations of hydroquinone as indicatccl (9A 0). Samples rrcrc introduced into the spcctrophotomctcr flow ccll. cqullihrntcd at 37" C in 10 s. and absorbmcc nionitorcd f o r IO min.Carricr h u h alonc wiis iiddcd IO conlrol wnplcs ( 0 ) .ILadlngs werc corrcctcd lor rcaycnt-huffcr ;ihsorh;incc ii I i i R. D. Irons and D. A. Kcptun To determine whether hydroquinone i1)terfercdwith tubulin plymcrization in 3 similar manner. the effects of hydroquinone on colchicinc-tubulin binding were examined (Fig. 3). The binding constant (KJ for colchicine-tubulin was found to be 1.54 X 10' I h o l which is in good agreement with that (2 X- 10' ]/mol) reported for chick embryo brain tubulin (Wilson et ai. 1Y74). Hydroquinone had no effect on either the affinity or the number of colchicine binding sites on tubulin. TCBA was found to decay in a first-order manner as previously described (Wilson et al. 1974; Weisenberg et ai. 1968). Addition of excess GTP. which is normally bound to the tubulin molecule in a molar ratio of 1: 1 (Wilson et al. 1974), stabilized TCBA; however, hydroquinone markedly accelerated the decay of TCBA (Fig. 4). Hydroquinone interfered with the GTP-dependent stabilization of TCBA in a dose dependent manner (Fig. 5 ) . Equimolar I I ! I ! I 100 9 . I .. U 3 HYORGQUINONE COLCHICINE 1 10 CONCENTRATION ( M I % 10' Fig. 2. Concenrration-response cunes for colchicine and hjdroquinone inhibition of microtubule polymerizarion. Inhibition of polymertzaiion was calculaied 3s % of control (ordinate) afrcr 10 min incub.mon .VI 31- C for diffcrcnt conccntrations (.hscirsa) of colchicinc ( 8 )o r h?droquinoric ( 0 ) 1-001 I i i I Effccrs o i Benzene conccntrations c rclati\e to contr lowest concentr; of TCBA incre: incrcase of ['"C: Incubation unde TCB.4 decay (Fi tubulin and the ['"C]hydroquino ATP in PEG b chrornatograhpy radioactivity app that of the nuc catcchol. hlasimurn rr parentheses) we .2 6 10 5 Fig. 3. Scatchard plot of colchicinc binding IO purificd rat ttihulin. The binding of colchicine to tubulin ( I inyml) 120 min a h incubation 31 37'C d o n c ( 0 )or in rhc prescnce of 2 x IO-' 51 hydroquinonc ( 0 )was cxamincd for various conccntraiions of colchicine. 'the m o h r r3Oo of Ilydroquinunc. io rubulin H'J\ 23: 1 I Fip. 5. Conccntratioc I dcca! :]I iiirying con Purifird tubulin preF hl I:H]colchicine (0. 37'C for 3homin in concrnir;itions of G? ( 0 , rhc addition of ( S ) .caicchol I.) or mc;m 2 SE for 3 (11 cxpcriincnrr 1 1 1 ? D.A . Scrtun trizati6n in ;I .inding \vcrc' :n \vas found x 10" Ihioi) .quinone h ; ~ d iing sites on ,ly described TP. which is M'ilson et A. derated the ?-de pendcn t . Equimolar I Effccts of Benzcne 'Metabolites on Microtubulc Polymerization 301 concentrations of GTP and hydroquinone resulted in a 50% decrease in TCBA. relative to control preparations. Phenol and catechol were effective only at the lowrestconcentration of GTP. The effect of hydroquinone on GTP stabilization of TCBA increased with time (Fig. 4) which correlated with the first-order increase of [''Clhydroquinone binding to tubulin with time (data not shown). Incubation under nitrogen completely inhibited the effect of hydroquinone on TCBA decay (Fig. 6). To distinguish between a direct effect of hydroquinone on tubulin and the formation of a hydroquinone-nucleotide complex. 0.4 m M [lJC]hydroquinone(2 pCi) was incubated for 2 h with 7.6 m M GTP or 6.9 m M ATP in PEG buffer at 37C. Separation of respective fractions by paper chromatograhpy revealed no "C-label migrating with GTP or ATP, all radioactivity appearing as a single peak with a mobility approximately one half that of the nucleotides. Identical results were obtained with phenol and catechol. Maximum molar ratios of hydroquinone: tubulin (ratios are given in parentheses) were calculated for concentrations of the benzene metabolite pome curvcs pinonc ! of lated as 5 r 10min iffeerent of colchicinc rot tubulin. min after < IO-' hi ions of was 13: 1 Fig. 4. Effect of hydroquinone and GTP on TCBA. Purified tubulin containing 2 x M 13H]colchicine (0.1 pCi). was incubated in the prcsencc of 2.5 mM GTP (0).absence . of GTP (a) or in the absence of GTP with Z x IO-' Xi hydroquinone (0) - ' 0 I20 240 360 4 2 0 INCUBATION TIME (MIN) s".ilOOr * VJ Fig. 5. Concentration-response curves for TCBA decay at varying concentrations of addcd GTP. Purificd tubulin preparations. containing 2 x M [?HIcolchicine (0.1 uCi), werc incubated at 37"C for 360 rnin in thc presencc of varying conccntrations of GTP (abscissa); with or without ( 0 )the addition of 2 X IW' M hydroquinone (0).catcchol (m) or phcnoi fA). Values present mean 2 SE for 3 (trcatcd) and 4 (control] experiments *- I -T , I I I I I I 01 IO 10 CONCEFITRATION CTP ADDED (MI#10' i ii I ! I i ! I I I +" 302 K. D. Iron\ and D.A. Scptun f:lIcuI\ of Bcr 1I Control 120 -- ~ lydroqutnone Catechol. Fix. 6. Eflcct o f anacrohic conditions oii Iiydroquinonc accclcration of TCUA dcciiy. Purified tubulin 1 I esposure (1 bcnzene arc ar,d 4) hv prcpar;itions. containins 2 x lU-b h.l ['Hlcolchicine (0.1pCi) and 1.3x I Distinguishi 1O-'M GTP. were incubated at f of benzene f 3 P C for 360min with 2 x 10-'M hydroquinone. catechol or phenol 1 \vi th tubulir Hydroqr I40 (data not shown) in air G )or under Nz ( Z ) . Carcchol or phenol had no effect on TCBA either in air or the affinity irreversibly i20 undcr N:. Values reprcsenr mean C binds reverr for 3 expcrimcnts. * Significantly diffcrent from control ( p < 0.006) i effects rep0 hydantoin c iI appears to t tubulin poly not understl 1 (Kuriyama L (\\'ikon et a . I-. Fig. 7. Electrophoresis of purified riit brain tubulin Rat brain tubulin (300 ug) was incubated for 1 h decay is irrc. 1 a functional 1 1978). and n> I 10 n V6 2 DF in the prcsencc of 2 x lo-' hl 1"CJhydroquinone (0.5 pCi) and subjcctcd to SDS polycr$wnidr disc gel electrophoresis on 8 cm polyacrylamide (7.5%) gels. Counting gcls wcrc CUI into 0.5cm slabs. solubilizcd and radioactivity ( 01 dclcrminrd by liquid scintillation. Parallcl gels wcrc stained with Coomassic Bluc and dcnsitornctric profiles ( - - - ) obtained bv scanning at 525 n m using a Helena dcnsitomcicr. Sligh molecular weight (HXl\V) Irxiion: low niolcculx wight (TU) fraction: and dyc front IDF) ilrc indicutcd. ! glycerol. apF 11 (hlellon and has been we1 binding sites 1 t u n -SHgrC izution at rat et nl. 1978). 1 groups are ohxrvcd u: I H! droquino, FRACTION tubulin mola GTP bur aF which produced the following effccts: 50% inhibition of tubulin polymerization (0.67: 1); 100% inhibiton of tubulin polymcrization (3.3: I ) ; and 50% incrcasc dcpendcnt IT none: tubuli iiccclcration in TCBA decay (2.3: 1). Elcctrophoresis (Fig. 7) revealed the majority of [14C]hydroquinone-associatedradioactivity to be bound to the high molccular weight fraction of tubulin-associated proteins. previously shown to activel! incorporatc 32P in vivo (Lagnado and Kirasov 1975). number ofh \\it h tubulin. 1 associated n I The depi marked difi. provides eyi Discussion H! droquino: phcnol and A comparison between the effects of benzene and colchiciric rcveals thc I reactive imer following similarities: 1) exposure to high doses in vivo results in a Gz'Mblock in I eifects of qu proliferating cells in bone niarrow (hluirhcad et al. 1980); 2) chromosomal tubulin inreg- abnormalities indicative of mitotic arrest are observcd following in vivo bcnzcncl involved in ! I L .. 2nd D. A. Seprun iaerohlc h x i i t i o n s xeleration of icd tubulin ning Z x I U - " % ~ .Ci)and 1 . 3 x incubatcd at ith 2 X 1O-'h1 hol or phcnol air ( Z ) or under . phenol had no her in air or .present mean 2 Significant1y 31 (p -< 0.006) ,t brain tubulin. m e d for 1 h .vdroquinone acrylamide acrylamide into 0.5 cm >) determined ere stained ric profiles n usir.g a r weight ht (TU) cated. olyrncrization 50% incrcasc c majority of igh molecular in to activcly 2 reveals thc G,/M block in chrorn osomd vivo bcnzenc Effects of Benzene hletabolites on hlicrot&ule Polymerization 303 exposure (Laskin and Goldstein 1977); 3) the principal hydroxyrnetabolites of benzene are mitotic inhibitors of cells in culture (Parmentier and Dustin 1953); I and 4) hydroquinone inhibits purified tubulin polymerization in vitro. Distinguishing benzene from colchicine are: 1)the requirement for metabolism of benzene for toxicity; and 2) differences in the mode of binding and interaction with tubulin between hydroquinone and colchicine. c Hydroquinone does not bind at the colchicine-binding site nor does it alter the affinity of colchicine at the ICjo of the former. Hydroquinone binds irreversibly to the high molecular weight fraction of tubulin whereas colchicine binds reversibly to the low molecular weight form (Wilson et al. 1974). The effects reported here for hydroquinone are similar to those reported for the hydantoin compounds (MacKinney et al. 1978); although, hydroquinone appears to be about 10-fo!d more potent than diphenylhydantion at inhibiting tubulin polymerization. The biochemistry of the TCBA decay phenomenon is not understood but it coincides with an irreversible loss of polymerization (Kuriyama and Sakai 1974) and evidently involves denaturation of the protein (Wilson et al. 1974). Unlike the dissociation of colchicine from tubulin. TCBA Jccay is irreversible. The integrity of free -SH groups on the tubulin molecule is a functional requisite for stability (Kuriyama and Sakai 1974; Ikeda and Steiner 1978), and agents which stabilize TCBA, such as guanine nucleotides or glycerol. apparently do so through interaction with -SH groups on the protein (Mellon and Rebhun, 1976; Mann et ai. 1978). The GTP-binding site on tubulin has been well characterized and involves 2 out of approximately 11titratable -SH binding sites on the protein (Mann et al. 1978; Iheka and Steiner 1978). These two -SH groups react with sulfhydryl reagents which inhibit tubulin polymerization at rates much higher than other -SH sites on the tubulin molecule (Mann et al. 1978). Colchicine-binding to tubulin is not affected until five or more -SH groups are blocked, and complete inhibition of colchicine-binding is not observed until all -SH groups are blocked (Iheka and Steiner 197s). Hydroquinone does not alter colchicine binding affinity at a hydroquinone: tubulin molar ratio in excess of 20: 1. Hydroquinone does not react directly with GTP but apparently interferes with GTP-tubulin binding in a concentration dependent manner and only under aerobic conditions. The ratio of hydroquinone: tubulin concentration accompanying a loss of tubulin polymerization or acceleration of TCBA decay is consistent with a reaction involving a limited number of binding sites (1-3) and is indicative of a relatively specific interaction with tubulin. This interaction presumably involves the oxidation of -SH groups associated with GTP-tubulin binding. The dependence of hydroquinone reactivity on oxygen, together with the marked difference in potency of hydroquinone versus catechol cr phcnol. provides evidence for an oxidative mechanism of action involving tubulin. Hydroquinone spontaneously oxidizes under physiologic conditions whereas phenol and catechol require further enzymatic oxidation in order to form reactive intermediates (Greenlee and Bus 1980; Mason 1979). Assessment of the effectsof quinonc and rclated compomds. such a hcnzoquinonc. on purified tubulin integrity and binding offers a n in vitro modcl f o r thc itudy of mcchanisnis involved in frcc radical interaction with ~ i i i ~ r o n i ~ l c c u l c ~ . I i ! I I ! 1 ! I I I ! . 30.1 R. 11. Irons and 0.A. Ncptun In addition to causins mitotic arrest in culture. hydroquinone inhibits mitogen stimulation of lymphocyte growth at non-cytotoxic conccntrations (Wierda et al. 1980). and has been shown to retard tumor growth in mice receiving melanoma transplants (Chavin et al. 19SO). Agcnts which modulate cytoskeletal structure (e.g. inhibitors of microtubule poiymcrization) inhibit mitogen stimulation of lymphocytes (McClain and Edelman 1980) and alter ccll surface reccptor mobility (Edelman 1976). Although the functional role of microtubules in the control of these cellular processes remains to be elucidated. the interference of hydroquinone with microtubulin integrity and nucleotide binding may have broad implications for the study of the mechanism of benzene toxicity at the molecular level. References I i ! i Borisy GG, hlarcum. Jhl. Olmstead JB.hlurphy DY. Johnson KA (1975) Purific;ition of tuhulin and associated high molecular weight proteins from porcinc brain and char;ictcrizatiun of I I rnicrotubulc assei~~hliyn vitro. Ann N Y Acad Sci 253: 107- 131 Borisy GG. Olmsrcd JB. 3larcum Jhl. M r n C (1Y7-1)Microtubulc asscinhly iii vitro. Fcd Pruc 33: 167- 174 Bryan J (1971) Biochemical properties of microtubules. Fed Proc 33: 152-157 Chavin W. Jelonek EJ Jr. Rcccl AH. Binder LR (19SO) Survival of.micc receiving me1;inoinii II transplants is promoted by hydroquinone. Science 20s :JUS-410 Dcysson Ci (1975) Xlicrotubules and antimitotic substanccs in microluhulrs and microtubule inhibitors. Borgcn hl. DcBrabanJer M (cds) Elsevicr. North llolland NCW York. pp 427-451 Edeiman GN (1976) Surfacc modulation in ccll recognition and ccll growth. Sciriicc I i 192: ?.18-219 Gaskin F. Cantor CR. She1;insLi XI1. ( ( 197.5) Turbidinictric studies of [be iii vitro ;iswnlily ;inti I disassembly of porcine ncurutuhulc\. Aiin NY Acad Sci 253: 133- 140 I Gornall AG. Bard;iill CJ. D;ivid A I A I (IY40) Dcterrnination o f w u m prowin5 by incans of thc hiurci reaction. J niol Chcm 177:751 Grccnlrc WF. Bus JS ( IOSII) A p r o p o d mcchmisni fur hcnzcnc tcisicity: Formation of rc;ictiw intcrmcdiatcs from pelyphcnul mct;lboiites of bcnzcnc. Ph;irm;iculogist 22 :-:S9 Himcs RH. Himcs VB ( 1980) Inhibition of tubulin assembly by ethyldcctyl-acrylatc. ;I sulfhyiryl reagent and potential analog of cytochalasin A. Biochem Biophys Acta 621: 338-342 lkeda Y. Steiner M (1975) Sulfhydrylsofplatclc~tubulin: Their role in polymerization and colchicine 3 binding. Biochemistry 17: 3454-3459 Irons R D (1960) Benzene induced rnyelotoxicity: Application of flow cytofluoromerry for Ihr. evaluation of early prolifcralive changes in bone marrow. Environ Health Psrspcct (in press) Irons RD, HeckHd'A. hloorc BJ. Muirhead KA (1979) Effccts of short-term hcnzcnc administration on bone marrow ccll cycle kinetics in the rat. Toxicol Appl Pharmacol 51 :399-409 Katz NL (1972) Effects on fros ncurornuscular transmission of agcnts tvhich ncr upon microlubulc+ and microfilamcnts. Eur J Pliarmacul 19: 88-93 Kuriyama R. Sakai l i (IY71) Role of tubulin-SH group5 in polymerization to microlubulcs. J Biochcm 76: 651-651 i i Laccy PE. I Jowell SL. Young DA. Fink CJ (190s) Ncw hypothcsis of insulin hormone secretitin. Katurc 219: 1177-1179 Lagnado JR, Kirasov EP (197.5) Studirs on the phosphorylation of brain microtubule yru!c.cn and microtubule-associatccl lipids in microtuhules and inicrotutiulc inhihitors. Burger> .\I. DeBrnbanJrr X I (eds) Elbcvicr. North tIoll.ind. Nrtv York. pp 12?-\10 Laskin 5. Goldstein OD ( 19771 Hcnzcne tosicicv: A critical craluiition. J . 'Jc~'(ic01.Envirun ~~cJI:!I (Suppl) 2 : IJX I L ! mct\ u X1;icliinncy microrut- Slnnn K. Gi and char Leu 92: Slaron RP ( Rcv Bio S1cCl;lin D; agents t: Xlisumi 51. . interact1 Slellon M G 70: 226- Sluirhead K cwlustic Parmcntier hydroqt Shapiro .U elcctroF Tragdnos F dihydro mamma Weisenberg its rrlai U'ierda. D. micc. P \Vilson L. microtu \\'ilwn L. ,' urchin Z\\cic ti. . I<ccci\.ed ; 6 r7d D. A. 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