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&CStbRmIlbrPrJdw-thu~~dty~fibu~amMedldc.atCr E fb Inhibition of Lymphocyte Transformation and 3' Microtubule Assembly by Quinone Metabolites of Benzene: Evidence for a Common Mechanism Richard D. Irons, Douglas A. Neptun and Richard W. Heifer Department of Pathology Chemical Industry Institute of Toxicology Research Triangle Park, NC 27709 Received March 24, 1981 Hydroquimne inhibits microtubule nssembly and suppresses lectin-induced transformation of lympboeytes. Other workers have established a role for microtubules in modulating cell respow to blastogenic stimuli. Several lines of evidence suggest that a) suppresioo of lymphocyte activationby hydroquinoneis mediated through interfennee with the cytoskela ton, specifknlly, Inbibition of microtubule function, b) insctlvatioo is an SH-dependent process, and e) p b e m q u i w n e is the most likely molecular species responsible for these events. INTRODUCTION Benzene is an important industrial chemical which is toxic to bone marrow and g lymphoid cells. Repeated exposure to benzene in experimental animals results in a3 lymphocytopenia (23, 24, 41, 52, 581, a decrease in bone marrow cellularity (23), alterations in cell cycle kinetics of proliferating bone marrow precursor cells, and chromosomal aberrations (23,27,40,53). Toxicity to bone marrow and lymphoid 5 ic organs correlates with the concentration of the 2 principal dihydroxy metabolites of 3 benzene accumulating in these tissues, hydroquinone (HQ)and catechol (CT) (17, 5 18). We have recently reported that HQ inhibits lectin-stimulated lymphocyte 1" activation in culture (44)and interferes with microtubule assembly and tubulin-col- chicine-bindingactivity (TCBA)in vitro (25). Both of these phenomena appear to be aF F mediated through interaction with sulfhydryl (SH)groups: in the first case, with intracellular SH groups critical for lymphocyte agglutinationand blastogenesis, and :: in the second, with SH groups necessary for maintaining microtubule functional integrity. An impressive body of evidence suggests that these 2 processes are 6 directly related. Microtubules, microfilaments, and their associated proteins are known to be involved in the regulation of cell surface receptor movement and cell growth (IO, E 36). Lectin-stimulated lymphocyte activation is a microtubule-dependent process; microtubule-disruptive agents enhance lymphocyte blastogenesis at low concentrations (43, 51.61) but inhibit mitogenesis at higher concentrations (10, 16, 19,46, 359 I I iI jI ! I I 360 IRONS ET AL. 51,56). SH group-specific reagents, such as N-ethylmaleimide(NEM), cytochdasin A, 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), and p-chloromercuribenzenesulfonate, inhibit microtubule self assembly and TCBAin vitro (20.21,28,38). Of these reagents, those which penetrate the cell membrane (NEM, cytochalasin A) disrupt a variety of immune cell functions requiring cytoskeletal-dependent conformational change ( I 1, 14,551. Colchicine must also enter the cell to affect motility and surface receptor movement (2). The mechanism of SH involvement in the self assembly of microtubules is unknown; however, studies have established the importance of free thiol groups in mahtaining the functional integrity of microtubules (22,28,33,38). Purified tubulin contains between 8 and 11 titratable SH groups, interaction with 2 or more ofwhich results in inhibition of microtubule assembly (22, 28, 33, 38). Mann et a/. (33) reported that 2 SH groups demonstrate statistically greater reactivity with NEM, a SH-alkylating reagent, than other free thiol groups on the protein and that the identical SH groups are associated with GTP binding to the tubulin molecule. Studies in this laboratory have demonstrated that HQ inhibits microtubule self assembly and enhances the decay of TCBA in vifro (25). Addition of GTP amelio- rates the latter effect in a concentration dependent manner. Hydroquinone and p-benzoquinone (p-BQ), the oxidation product of HQ, sup press lectin-stimulated lymphocyte blastogenesis in vitro at noncytotoxic concentrations ( p M )and, under physiologic conditions, HQ autoxidizes to form p-BQ, presumably forming the semiquinone radical in the reaction (26). In this scheme, the suppression of lymphocyte activation or cytotoxicity could be mediated through the formation of superoxide anion,p-semiquinone or direct alkylation byp-BQ. The SH reactivity of the quinones is well known (39) and has been demonstrated in the context of reactions involving 6-hydroxydopamine and related compounds (5, IS, 31, 48). In this paper, the effects of the polyhydroxy-metabolites of benzene on microtubule assembly are characterized and correlated with their effects on cytoskeletal-dependent lymphocyte activation in culture. A molecular mechanism is proposed for the modulation of lectin-induced lymphocyte blastogenesis by quinone compounds. MATERIALS AND METHODS Isolation of Rat Spleen Lymphocytes. Spleens pooled from at least 4 male Fischer-344 rats (Charles River) 6 to 8 weeks old were minced, homogenizedgently with a loosely fitting Teflon homogenizer, and filtered through nylon mesh in a volume of 20 ml RPMI 1640 medium (GIBCO) supplemented with 7.5% fetal calf serum (FCS) (Kansas City Biologicals). AI1 tissue, cells, and media were handled aseptically and kept on ice. The cell suspension was centrifuged at 300 xg for5 min, and the pellet was resuspended and washed once with medium. Isopycnic sedimentation of cells on Ficoll-paque" (Pharmacia Fine Products)was achieved by centrifugation at 400 x g for 30 min at 20 C; lymphoid cells at the interface were removed by aspiration and washed twice. Exposure and Stimulation with Mitogen. Exposure of cells to varying concentrations of metabolites or reagents was carried out by preincubation at 37 C in phosphate-buffered saline (pH 7.2). Thoroughly washed cells were plated at a density of 2 x IO5 cells per well in 96-wel1, flat bottom microtiter plates (Bellco) containing 0.2 ml RPMI 1640 medium supplemented to final concentration with F ,,,ltCJ ethanol ing of 5 tinin (P was as: New Er filter Pa were e. -d . cse Individl represe vidual c Isolat from mthis ma polyme +l<ewhr iM L!bilS PI turation ity (log criteria TCBA describ reagent -of vario ';\tS v e ai design. using C Spect protein 288 nm using a against COlCf 'P. t I 3Q.L Effec Hydroa biphasil less tha 'pM SUJ Y 10- yi INHIBITION O F LYMPHOCYTE TRANSFORMATION 361 7.5% FCS, 2 mM L-glutamine, 100 idml penicillin, 100 p g m l streptomycin (as a mixed supplement from Microbiological Associates), and 5 x M 2-mercaptoethanol (Sigma). Plates were incubated at 37 C in a humidified atmosphere consisting of 5% C02 95% air. Optimal blastogenic response to 5 pglml phytohemagglutinin (PHA)(Burroughs Wellcome)occurred on day 3 of culture. Synthesis of DNA was assayed by adding 0.2 pCi of [3H]thymidine(specific activity, 6.7 Ci/mmol; New England Nuclear) per culture 6 hr prior to harvest. Samples were collected on Titer paper, dried, and counted by conventional liquid scintillation methods. Cells were examined by phase contrast microscopy, and an agglutination index was scored on a scale from 0 to 4 + (44). 0 indicated no evidence of aggregation; 1 + represented the presence of clusters containing less than 50 cells interspersed with individual cells and was encountered in cultures containing no mitogen; 2+ to 4+ represented progressively larger aggregates containing blast cells with fewer individual cells. Isolation and Characterization of Rat Brain Tubulin. Brain tubulin was isolated 4. from male Fischer 344 rats by the cycle procedure (6,7,25,47).Tubulin prepared in 5 this manner was stored at -80 C and used within 2 weeks. Temperature-dependent polymerization at 37 C was measured turbidometrically at 350 nm as described elsewhere (12, 13). Tubulin polymerization experiments were conducted using 20-mM phosphate buffer (pH 6.92). Measurement of polymerization under conditions producing greater than 80% inhibition were not reported due to partial denaturation of the tubulin. Denaturation was determined to be significantwhen turbid1 ity (log absorbance plotted versus the log of the wavelength) failed to fulfill the criteria of the Raleigh-Gans approximation for long thin rods (4, 12,54). Decay of TCBA was determined by using [3H]colchicine(1 Ci/mmol) and gel-filtration as described elsewhere (25) except substituting 20-mM phosphate buffer. The pH of reagent and assay solutions was maintained at 6.92 T'- 0.02. In assessing the effects of various treatments on tubulin polymerization or TCBA decay, replicate experiments were conducted on different batches of tubulin and on different days. Results were analyzed by 2-way analysis of variance using a randomized complete block design, and mean differences were evaluated for significance at thep = 0.05 level using Duncan's multiple range test (50). Spectral Analyses. The stability of p-BQ and HQ in the presence or absence of protein was compared by measuring changes in their absorption maxima (248 and 288 nm, respectively) with time. Spectral measurements were conducted at 37 C using a Cary model 219 scanning spectrophotometer. All readings were blanked against buffer or the addition of protein. Colchicine, CT, L-cysteine (CYS), dithiothreitol (D'IT), DL-lysine, L-serine, GTP, NEM, and tyrosinase (EC. 1.14.18.1) were purchased from Sigma. HQ, p-BQ, and 1,2,4-benzenetriol (1,2,4-BT) were obtained from Aldrich. RESULTS Effect of HQ and BQ on kctin-Induced Activation of Lymphocytes in Culture. Hydroquinone modified PHA-stimulated DNA synthesis in lymphocytes in a biphasic manner (Table 1) as assessed by [3H]thymidineuptake. Concentrations of less than I p M enhanced PHA stimulation while concentrations of greater than 1 /AMsuppressed mitogen response. Enhancement was optimal at approximately 1 x lo-' M HQ and became progressively smaller at lower concentrations where it i I 1 362 IRONS ET AL. NEM - *Cell. stimular more f h PHA-st conrrol penetr respon or NE' depenc c uIt urc 363 I 364 IRONS ET AL. CONCENTRATION IL M) Fig. I. Log concentration-inhibition curves for (A) p-BQ, (B) HQ, (C) NEM, or (0)I,2,4-BT on purified tubulin polymerization in vitro. Inhibition of polymerization was calculated as the percent of control for incubation with different concentrations of inhibitors. response via interaction with intracellular SH sites. Effect of Benzene Metabolites on Tubulin PoIymerization. Tubulin polymerization i putative oxidation product of CT and has been shown to autoxidize in a reaction analogous to HQ (26). Polymerization was inhibited by p-BQ, HQ, 1,2,4-BT, or NEM in a concentration-dependentmanner (Fig. 1). The linearity of a semi-logarithmic plot of inhibition compared with the concentration of p-BQ or NEM is characteristicof a first order reaction and is suggestiveof a unimolecularprocess in which p-BQ and NEM react directly to inhibit tubulin polymerization. Minimally effective concentrations ofp-BQ and NEM were the same, although differences in the slope of the lines may reflect increased affinity for some SH sites by NEM relative to p-BQ. These results were contrasted by the nonlinearity exhibited for HQ and 1,2,4-BT inhibition of polymerization when plotted in the same manner. Linearity on a loglog plot would indicate that inhibition by these compoundscould be characterized as either second order or the sum of the products of 2 first order processes. A log-log plot of the data presented in Figures 1 B or D failed to yield a linear transformation, indicating that the kinetics of inhibition of tubulin polymerization by HQ and 1,2,4-BT are of a higher order of complexity. Catechol had no ' :I h P T fi tc TABLE 3 Effect of Tyrosinase on Catechol Inhibition of Tubulin Polymerization Catechol (1 x Tubulin Polymerization (% Control) MI Tyrosinase (10 &ml) Catechol + Tyrosinase 104 f 1.9. 94.7 f 0.9' 8 2 Zb .Mean 2 SD of 3 separate determinations. bMean 2 SD of 4 separate determinations. LY tu ah re; , th; sin p-! -i INHIBITION OF LYMPHOCYTE TRANSFORMATION 365 -210 240 260 280 mki 12 08 L Q )n of 04 `1 220 240 260 28U WAVELENGTH Inml -2 WAVELENGTH (nml n Fig. 2. Effect of protein on the autoxidation of HQ and the disappearanceofp-BQ under physiologic d conditions. (A) Decomposition ofp-BQ (0.05mM absorptionmaximum = 248 nm) in20-mM phosphate 1 buffer(37C.pH 6.92). (B)Disappearance ofp-BQ following additionofbovine serumalbumin(2 mg/ml). r (C)Autoxidation of HQ (0.2 mM, absorption maximum = 288 nm) and the appearance ofp-BQ. (D) Autoxidationof HQ in the presence of albumin. Numbersindicate the scan time (min) after the addition ofp-BQ or HQ. i 1 effect on tubulin polymerization at concentrations of 5 x IO-` M, a finding which was consistent with its failure to autoxidize appreciably under physiologic condi- tions (26). However, 1,2,4-BT,a putative enzymatic oxidation product of CT, was approximatelytwice as potent as HQ in inhibitingtubulin polymerization. 1,2,4-BT , has been demonstrated to autoxidize more rapidly than HQ (26). In order to further assess the role of quinones in the inhibition of tubulin : polymerization, CT was added to tubulin in the presence of tyrosinase (10 pg/ml). Tyrosinase converts CT to the o-quinone directly via 2 electron transfers, thus, formingno reactive intermediates in the reaction (34). Theo-quinones are reported 4: to be extremely electron-deficient species capable of reacting with external nucleophiles such as SH, amino or phenolic hydroxylgroups and have been shown to react 0' with SH groups in preference to other intramolecular reactions (48). In the tyrosinase-catechol system as little as 1 x M CT resulted in complete loss of tubulin polymerization and partial denaturation of the protein (Table3).Tyrosinase alone had no effect on tubulin polymerization. Consistent with its greater SH ' reactivity, o-BQ appears to be a more potent inhibitor of tubulin polymerization Y- than doesp-BQ. These findings suggest a direct benzoquinones-tubulininteraction i similar in nature to that observed with NEM. 8- Stability ofp-BQ and HQ in the Absence and Presence of Protein. Spectral scans of p-BQ and HQ in the absence or presence of albumin revealed that the rate of disappearance of absorption maxima forp-BQ normally seen with time was greatly accelerated by the addition of protein (Figs. 2 A and B). The rate of disappearance I I c 1 COI r- ( 27 dir bet evt der SH !' L17h intt add Pol: Fig Aero} were 'par cub $ INHIBITION OF LYMPHOCYTE TRANSFORMATION 367 '"I !. Fig. 4. Effect of various compounds on the acceleration ofTCBA decay byp-BQ or NEM. Incubation conditions were the same as those described in the legend to Figure 3 except that the concentration of .. GTP (2.5 x IO+ M)was constant throughout. Concentrations of DTT,lysine. serine,p-BQ. and NEM I were all 1 x IO-' M.Values represent the mean z SEM for 3 separate experiments. (9) Significantly '' different from control (P< 0.05). (1)Significantly different from DTT treated. (2,10, 11,36,37,55).Although there is not uniform agreement on the importance of direct cell-cell communication in the initiation of cell division, agglutination has been reported to be a requisite for blastogenesis and represents one of the earliest events associated with initiation of cell division (32, 57). Studies have repeatedly demonstrated that inhibition of blastogenesis by microtubule-disrupting agents or SH reagents does not involve alterations in lectin-bindingto surface receptors (3,9, 16, 4 3 , alterations in GSH concentrations (9, 30), or interference with energy production within the cell (8,30,35). Our findings provide additional confirmation that microtubule-disruptive agents inhibit cell agglutination and simultaneously interfere with density-dependent stimulation of cell growth (3, 36, 37, 44).In addition, these findings indicate a) that suppression of lymphocyte activation by polyhydroxy-metabolites of benzene is mediated through inhibition of microtubule i Fig. 5 . Effect of anaerobic conditions on acceleration of TCBA decay by I .2.4-BT, p-BQ, or NEM. Aerobic conditions were the same as those described in the legend to Figure 4. Anaerobic incubations were conducted using degassed solutions saturated with Nz.Values represent the mean t SEM for 3 separate experiments. (5) Significantly different from control. fl) Significantly different from same incubation in air. (11) Significantly different from anaerobic control. 368 IRONS ET AL. assembly, b) that both processes are SH-dependent. and c)that benzoquinones are the most likely molecular species responsible for direct interaction at SH-depen- dent sites on microtubules. p-Benzoquinone, the autoxidation product of HQ, is more potent in both systems: the metabolites suppress both lymphocyte response and microtubule assembly at the same concentrations. N-ethylmaleimide, an SH- alkylating agent, mimicked the effects of benzene metabolites, and sulfhydryl compounds, provided protection against the effects of both N E M and the metaboliteson lymphocyte activation and microtubule function. Pretreatment with a variety of compounds demonstrated the dependence of these processes on specific intracellular SH groups. Analysis of kinetic and spectral data suggests a molecular mechanism in which microtubule-associated SH groups demonstrate particularly reactive nucleophilicity toward quinones or semiquinones. Concentrations of HQ orp-BQ ( I x 10-*to 1 x 10-3M) which suppress lymphocyte Bactivation in culture are the same as those producing inhibition of tubulin polymeti- zation in v i m . Inhibition of lectin-stimulated blastogenesiscoincides with the loss 2 of cell agglutination. Only a small portion of the decrease in [3H]thymidineuptake observed following 14-hrpreincubation with HQ orp-BQ can be accounted for by "icell loss. Furthermore, we have reported other experimentsin which incubation for ,, 30 min with the same concentrations of HQ andp-BQ results in a complete loss of mitogenic activity in the absence of cell loss or adecrease in energy production (44). Complete protection against the suppressive action of HQ orp-BQ on lymphocyte activation is afforded by CYS but not lysine or serine. NEM inhibits blastogenesis - and agglutination at the same concentrations asp-BQ which is protected against by 3 CYS, whereas DTNB has no effect on either lymphocyte activation or agglutina- tion. These observations are indicative of an interaction with intracellular SH groups critical to lectin-stimulatedlymphocyte activation. Analogous observations with respect to SH specificityhave been presented for the effects ofp-BQ on tubulin stability in vitro. We have recently reported that HQ covalently binds to tubulin (25). The ratio of HQ:tubulin concentration which is attended by inhibition of polymerization is consistent with an interaction involving a very small number of binding sites. r Observations presented in this paper implicatep-BQas the ultimate toxic species in a reaction scheme in which HQ is autoxidized to p-BQ, although due to the circumstantial nature of the evidence one cannot entirely rule out the possibility that thep-benzosemiquinone intermediate is active as well. In either case proposals for the chemical reaction scheme betweenp-BQ orp-benzosemiquinone and tubu- lin are consistent with conjugate addition in which a highly polarized unsaturated carbon-carbon bond is subject to attack by a reactive thiol group acting as a nucleophile (39, 49). Identical models for thiol alkylation are provided by other ' inhibitorsof tubulin polymerization. namely NEM and cytochalasin A (20,29,30). The denaturation of tubulin by CT following the addition of tyrosinase suggests a greater reactivity of the 0-BQ. This observation is supported by studies of the oxidation products of 6hydroxydopamine in which the o-quinones possessed greater SH reactivity than did the p-quinones, based on their ability to inhibit SH-dependent enzymes. The demonstrated reactivity of p-BQ with tubulin ' suggeststhat tubulin may represent a more sensitive indicatorof SH reactivity than do enzymes such as CT-o-methyl transferase or calf thymus DNA polymerase ( 5 , IS). 1 1 1 r es are epen- {Q. is wnse 1 SH- vet. ' .Lh s on Sts a trate cyte ienloss . ior s of 14). Yte :sis by na;Y of is JS. in le tY d a r 1. J I INHIBITION OF LYMPHOCYTE TRANSFORMATION bmlW o-@ I I OH 369 I+ I 1 Fig. 6. Schematic diagram of primary benzene metabolism resulling in the production of potentially lymphotoxic metabolites. (*) Putative or demonstrated alkylating activity toward intracellularnucleophilic groups. Depending on the endpoint used to measure toxicity, benzene metabolites may present with varying orders of potency. Hydroquinone is a more potent inhibitorof lymphocyteactivation than is CTin vitro (26,44)andin vivo (59).On the otherhand, CT is more potent at producing sisterchromatid exchange in human lymphocytes in culture (40). Such discrepancies could arise from digerences in the oxidation products of HQ and CT. We propose a reaction scheme in which HQ and CT can proceed along parallel or distinct oxidative pathways (Fig. 6). Hydroquinone spontaneouslyoxidizes to formp-benzosemiquinone andp-BQ. Catechol does not undergo appreciable autoxidation under physiologic conditions, but it could serve as a substrate for a variety of enzymatic reactions. Catechol can be oxidized to 1,2,4-BT which undergoes a similar autoxidation reaction to HQ, presumably forming the 2-hydroxy-analogue of p-BQ. Alternatively, CT may be oxidized by tyrosinase or a variety of oxidases to form potentially reactive products such as phenoxy-radicalsor o-quinones which would be expected to react indiscriminately with a variety of nucleophilic groups on protein or DNA in addition to SH groups. The relatively weak alkylating ability of the p-quinones could result in specificity forparticularly nucleophilic SH groups within the cell and account for their ability to interfere with cytoskeletal function in the absence of cytotoxicity. The observa* hat quinone metabolites interfere with normal cytoskeletal function suggestsa -=ism to explain bone marrow and immune suppression associated with Z+ GiZoenzene exposure. 370 IRONS ET AL. ACKNOWLEDGMENT I The authors thank Dr.Ronald P. Mason of the National institute of Environmenlal Health Sciences and Mr. Robert 0. Beauchamp for helpful discussions. REFERENCES I. Ando. Y. and Steiner, M. Sulfhydryl and disulfide groups of platelet membranes: determinationof sulfhydryl groups. Biochim. Biophys. Acta 311, 26, 1973. 2. Aubin. J. E.. Carlsen, S. A. and Ling. V. Colchicine permeation is required for inhibition of concanavalinAcappinginChinese hamsterovarycells. Proc. Nail. Acad. Sci. U S A 72,4516,1975. 3. Berlin, R. D. and Ukena. T. E. Effect of colchicine and vinblastine on the agglutination of polymorphonuclear leukocytes by concanavalin A. Nature New Biol. 238, 120. 1972. 4. Beme. B. J. Interpretation of light scattering from long thin rods. J. Mol. Biol. 89, 755, 1974. 5. Borchardt. R. T. Affinity labeling of catechol u-methyl transferase by the oxidation products of Chydroxydopamine. Mol. Pharmacol. 11. 436. 1975. 6. Borisy, G. G.. Olmsted, J. B.. Marcum. J. M. and Allen, C. Microtubule assembly in virro. Fed. 3 Proc. 33, 167, 1974. 7. Borisy. G. G., Marcum, J. B.. Olmsted. J. B., Murphy, D. B. and Johnson, K. A. Puriticationof tubulin and associated high molecular weight proteins from porcine brain and characterization of microtubule assembly in virro. Ann. NY Acad. Sci. 253. 107. 1975. 8. Chakravany. N. and Echetebu, 2.Plasma membrane adenosine triphosphatases in rat peritoneal mast cells and macrophages - therelation of the mast cell enzyme to histamine release. Biochem. Pharmocol. 27, 1561, 1978. 9. 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The action ofcytochalasin A on thein vitro polymerization ofbrain : .' tubulin and muscle G-actin. J . Suprumol. Srrucr. 5. 81, 1976. 21. Himes. R. H. and Himes. V. B. Inhibition of tubulin assembly by ethylace~ylacrylate.a sullhydd reagent and potential analogue of cytochalasin. Biochim. Biophys. Acta 621, 338, 1980. 22. Ikeda, Y. and Steiner. M. Sulfhydryls of platelet tubulin: Their role in polymerization and colchicine binding. Biochemistry 17, 3454, 1978. 23. Irons. R. D.. Heck, H. d'A.. Moore, 8 . J. and Muirhead. K. A. Effects of short term benzene administration on bone marrow cell Cycle kinetics in the rat. Toxicol. App/. Pharmacol. 51, 399, 1979. 24. Irons. R. D. and Moore, B. J. Effect ofshon term benzeneadministration on circulatinglymph~yte INHIBITION OF LYMPHOCYTE TRANSFORMATION 371 subpopulations in the rabbit: evidence for a selective B-lymphocyte sensitivity. Res. Commun. . Chem. Pathol. Pharmocol. 27, 147. 1980. 25. Irons. R. D. and Neptun. D. A. 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