Document YGqaxOvj1x9rDnpoXen2eg46V

CANCER RESEARCH 45,2471-2477. June 19851 Sister Chromatid Exchange Induction in Human Lymphocytes Benzene and Its Metabolites in Vitro' hlb-h- - Awrnry;r,dYY S- r-eaoIrv-L..Erexson.' James L. Wilmer, and Andrew D.Kligerman3 H e 1 R I ) J t a E&UY d l ~m.m&kOoa V n t a d J Sl& Msdkrd C g k r Iepartmentof Genetic Toxicology. Chemical Indusfry InsfifUteof Toxicology, Research Triangle Park, North Carolina 2770 RBSTRACT mas are the predominant neoplasias found in humans exposed to benzene, other types pf cancers such as Zymbal's gland and Previous in vivo studies have shown that low-dose benzene hepatocellular carcinomas are found more often in rodents ex- exposure (10 to 28 ppm for 4 to 6 h) in mice can induce sister posed to benzene(7-10). Great variability exists in the sensitivity chromatid exchange (SCE) in peripheral blood B-lymphocytes of individuals to benzene exposure. Factors such as age, geno- and bone marrow as well as micronuclei in bone marrow poly- type, immunocompetence,and life-style complicate assessment chromatic erythrocytes. Because benzene is metabolized to a of the probable causes of benzene toxicity (11, 12). Also, the variety of intermediate compounds and two of these, catechol length of exposure to and dose of benzene makes the interpre- and hydroquinone, have been reported to be potent SCE-induc- tation of epidemiological studies difficult (13, 14). Thus, the ers, it is possible that other known and proposed metabolites effects of low-level exposure to benzene remain an important could have chromosome-damaging effects in lymphocytes. In- although controversial issue (15-17). duced SCE frequencies, mitotic indices, and cell cycle kinetics Benzene is thought to be metabolized through a benzene were quantitated in human peripheral blood T-lymphocytes ex- oxide intermediate by cytochrome P-450 in the liver (la), and posed to benzene, phenol, catechol, 1,2,4-benzenetriol, hydro- most of the benzene oxide spontaneously rearranges to form quinone, 1,4-benzoquinone, or frans,trans-muconic acid. Three phenol (19) (Chart 1). Most of the phenol is conjugated and proposed metabolites of phenol, 4,4'-biphenol, 4,4'-diphenoqui- excreted, but further ring oxidation can form hydroquinone(20), riorie, and 2,2'-biphenol, which can be generated by a phenol- which spontaneously oxidizes to 1,4-benzoquinone.Also, ben- horseradish peroxidase-hydrogen peroxide system were also zene oxide is conjugatedwith glutathione by epoxide transferase examined. Benzene, phenol, catechol, 1,2,4-benzenetriol,hydro- to yield the inactivation product phenylmercapturic acid (21). In quinone, and 1,4-benzoquinone induced significant concentra- addition, lymphocytes could conceivably convert benzene oxide lion-relatedincreases in the SCE frequency, decreases in mitotic to benzene glycol by the action of epoxide hydrolase, which is indices, and inhibition of cell cycle kinetics. Based on the slope found in human lymphocytes (22). Dehydrogenationof the glycol of the linear regression curves for SCE induction, the relative can then yield predominatelycatechol and, uponfurther oxidation potencies were as follows: catechol > 1,4-benzoquinone > of the glycol, frans,trans-muconic acid (21). A small amount of hydroquinone > 1,2,4-benzenetriol> phenol > benzene. On an catechol is ring oxidized to 1,2,4-benzenetrioI, but most of the induced SCE per p~ basis, catechol was approximately 221 catechol is conjugated arid excreted (23). times more active than benzene at the highest concentrations Because benzene is metabolized primarily in the liver and the studied. trans,trans-Muconic acid had no significant effect on the sites of toxicity are principally in the hematopoietic tissues, cytogenetic parameters analyzed. 2,2'-Biphenol induced a sig- transport of benzene or its active metabolites by blood is re- nificant increase in SCE only at the highest concentration ana- quired. Further metabolisrnof benzene or its known metabolites lyzed, and 4,4'-biphenol caused a significant increase in SCE can occur in the bone marrow (24, 25). Studies of rodents frequency that was not clearly concentration related. However, injected with either [3H]-or [14C]benzenehave shown that a both 2,2'- and 4,4'-biphenol caused significant cell cycle delay rnetabolite(s) binds covalently to rat liver DNA (26) and mito- and mitotic inhibition. 4,4'-Diphenoquinone caused only a signif- chondrial DNA from mouse liver and bone marrow (27,28). ["CI- icant decrease in mitotic activity. These data indicate that in Benzene is metabolized and bound irreversibly to macromole- addition to phenol, di- and trihydroxybenzene metabolites play cules when incubated with rat liver microsomes in the presence important roles in SCE induction. Furthermore, the results sug- of a NADPH-generating system (19). Tunek et a/. (19) also found gest either that benzene alone can induce SCE or, a more likely that the metabolites bind predominately to microsomal protein possibility, that rnononuclear leucocyteshave a limitedcapability and to a lesser extent, RNA. They suggested that a metabolite Io activate benzene. of phenol rather than benzene oxide was responsible for the binding. Sawahata and Neal (29)proposedthat phenol is metab INTRODUCTION olized to 2,2'-biphenol and 4,4'-biphenol by myeloperoxidase in the borie marrow (Chart 1). They demonstratedthat horseradish Benzene is generally accepted as being a carcinogen in hu- peroxidase or bone marrow homogenate (free of mature eryth- mans (1-5) and rodents (6-10). Although leukemiasand lympho- rocytes) in the presence of hydrogen peroxide could metabolize . ... . . . ' This research was funded by the Chemical Industry lnslitute 01 Toxicology, a pivalely funded instilute. phenolto 2,2'-biphenol, 4,4'-biphenol,and 4,4'-diphenoquinone. Previous studies have shown that benzene induces SCE4in Presenl address: Environmental Health Research and Testing, Inc.. P. 0. Box mouse bone marrow (30, 31). Other investigators have failed to 12199. Research Triangle Park, NC 27709 To whom requeslstor repnnls should detect a statistically significant increase in SCE frequency in be addressed. . .. , .- .. 'Present address: Environmental Health Research and Testing, Inc.. P. 0.Box 12199, Research Triangle Park, NC 27709. 'The abbreviations used are: SCE. sister chromatid exchange; MNL, mononu- Received 1/21/85; revised 311 1/85: accepld 3/12/85 clear leukocyte;AIiH, aryl hydrocarhi hydroxylase; DMSO, dimethyl sulfoxide. I CANCEH RESEARCH VOL. 45 JUNE 1985 -I , - 4 BENZENE-INDUCED SCE IN HUMAN LYMPHOCYTES benzene benzene oxlde benzene glycol tran8,trane.-muconlc acid COOH cls,clr-muconlc acld 1 P1,4-benzosernlquinone hydroqulnone phenol ehydrogenaae LO" catechol 1.2.4-bsnzenetrlol 2-hydroxy-1.4-benzoeemlqulnonn a' a"----e"0 4,TB..aHOH - - 4- n*o . o-" -O-H* ~-o . 0k i i6_spoi;leneoutr tyrosinase \ apontaneoq * 1 I1.4-benzoquinone peroxldase 1,2-be&e 2-hydroxy- 1.4-benzoqulnone A0 0 2,2'-biphenol proposed 4,4'-biphenol 4,4'-diphanoquInone (9! ch in cc MI Lo (Fi 0.: c1 ob so us B Chart 1. Schematic diagram of the known and proposed metabolismof benzene. The figure is a modified and composite representationof the metabolic pathways for benzene as reportedby Ruschel a/. (21), Irons el a/.(52). and Sawahata et a/. (64). peripheral blood lymphocytes of humans after occupational exposure to benzene (32-34). However, mice exposed to benzene concentrations as low as 10 ppm for 6 h exhibited an increased SCE frequency in peripheral blood B-lymphocytesand increased numbers of micronucleated polychromatic erythrocytes in their bone marrow (35). These results suggest that benzene or its metabolites in low concentrations in vivo are responsible for the chromosome-damagingeffects. Morimoto and Wolff (36) found that catechol, hydroquinone, and to a lesser extent phenol induced SCE in phytohemagglutinin-stimulated lymphocytes. Benzene did not induce any in- crease in SCE but showed some cytotoxicity at 5 mM and inhibited growth at 250 mM. Morirnotoet a/. (37) showed that rat liver S-9 augmented SCE induction in human lymphocytes exposed to phenol, catechol, and hydroquinone during a 2-h pulse treatment at 40 to 42 h of 3-day cultures. In addition, Morimoto (38) reported that benzene induced SCE in human lymphocytes only after incubation of the cells in the presence of rat liver S-9. Therefore, to understand further the chromosome-damaging effects of benzene, SCE induction in human T-lymphocytes was investigated after exposure in vitro to benzene, 6 known metabolites of benzene (phenol, catechol, 1,2,4-benzenetrioI, hydroquinone, 1,4-benzoquinone, and trans,trans-muconic acid) and 3 proposed metabolites of phenol (2,2'-biphenol, 4,4'-biphenol, and 4,4'-diphenoquinone). The purpose of this study was to determine if human lymphocytes exposed 24 t i after mitogenic stimulation (G1-S phase) would be more sensitive to the genotoxicity of benzene and its metabolites rather than if chemical treatrnent was begun at the start of culture (Go-Gl). Because increased concentrations of AHH in MNLs are found after mitogenic stimulation and concurrent exposure to aromatic hydrocarbons (39-41), chemical treatment at 24 h would coincide with an optimal time for potential metabolisrn. MATERIALS AND METHODS Blood Processing and Lymphocyte Culture Technique. Heparinized whole blood (35 to 60 ml) samples were drawn by venipuncturefrom the same healthy adult male for all experiments to alleviate any possible donor-to-donor variability. No attempt was made in these experiments to survey the variability known to exist in humanAHH inducibility;rather. the relative potencies of benzene and its known and proposed metabolites were studied. Whole blood was processed on Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ) density gradients, and MNLs were cultured as described previously (42). Two to 4 MNL cultures/ treatment were established by inoculating 10' MNLs into 1.9 rnl of complete medium composed of RPMl 1640, 10% heat-inactivatedfetal bovine serum, 100 units of penicillin, and 100pg of streptomycin sulfate/ ml. and an additional 292 pg of L-glutamine/ml. T-lymphocytes were stimulated to divide with 8 pg concanavalin A/ml. The cultures were incubated at 37% in a humidified COPatmosphere for 24 h. 5-Bromo- 2'-deoxyuridine (5p ~ w)as added at 24 h. The test compounds were added separately to cultures over the following concentration ranges (in p):benzene (5 to 7000); phenol (5 to 3000); catechol (5 to 500); 1,2,4- benzenetriol(5 to 500); hydroquinone(5to 500); 1,4-benzoquinone(5 to 500); frans,trens-muconic acld (5 to 500);4,4'-biphenol (0.1 to 500); 4,4'-diphenoquinone (0.1 to 100); or 2,2'-biphenol (5 to 500). The cultures were harvested at 72 h following a 4-h treatment with demecolcine (1.35p ~ ) . Chemicals. Phenol (99+%), catechol (99+%), 1,2,4-benzenetriol CANCER RESEARCIi VOL. 45 JUNE 1985 7177 BENZENE-INDUCED SCE IN HUMAN LYMPHOCYTES (99%), hydroquinone (99+"/0), and 1,4-benzoquinone (98%) were pur- chased from Aldrich Chemical Company (Milwaukee, WI) and dissolved in RPMl 1640 medium. Benzene was obtained from Fisher Scientific Company (Raleigh, NC) and also dissolved in RPMl 1640. trans,transMuconic acid (Aldrich), 4,4'-biphenol (Sigma Chemical Company, St. LOUIS, MO), and 2.2'-biphenol (Sigma) were dissolved initially in DMSO (Fisher) and diluted with RPMl 1640 to achieve a final concentration of 0.33% DMSO. 4,4'-Diphenoquinone (Pfaltz and Bauer, Inc., Stamford, CT) was also dissolved initially in DMSO and diluted with RPMl 1640 to obtain a final concentration of 0.8% DMSO. The test chemical stock solutions were prepared and sterilized (except benzene and phenol) using 0.22-pm Millex-GSfilter units (Millipore Corporation, Bedford, MA). Because of their volatility, benzene and phenol were prepared and added to cultures immediately prior to incubation. All test chernicals were added to the cultures in 2 0 4 aliquots. Slide Preparation,Cytogenetic,and StatisticalAnalyses. The slides were prepared as described previously, coded, and stained using a modified fluorescence-plus-Giemsa technique (43, 44). Fifty seconddivision metaphases, 200 consecutive metaphases, and 2000 nuclei were analyzedfrom each treatment for SCE frequency, cell cycle kinetics, and mitotic index, respectively, unless noted otherwise. All cytogenetic data were tested for normality and then subjected to a one-way analysis of variance with the level of significance chosen as 0.05 (45). A onetailed Dunnett's multiple range test was used to compare the SCE frequency of each concentration of chernical tested to the concurrent control or pooled control if warranted (45, 46). Linear equations were Table 1 Effect of benzene and its known in vivo water-soluble metaboliteson the SCE hequency, mitotic activity, and cell cycle kinetics of human peripheral blood T-lymphocytes exposedin vitro The removalof blood, treatment of MNLs, culture of lymphocytes, harvest, and slide preparationwere as described in "Materialsand Methods." Fifty seconddivision metaphases, 2000 nuclei. and 200 consecutive metaphases were analyzed for SCE, mitotic index, and cell cycle kinetics. respectively, unless noted otherwise. ~__._ ~ __ _______ Cell cycle kinetics ("A) ___ Chemical RPMl 1640 control Concentration (#M) SCEs/metaphase 8 68 i 0 30'- Mit_otic_in_de_x (%) 5 99 f 042' First division 17 0 i 2 6' Second division 41.Of 5.7 Thud division 30.0 i 3 4 Fourth division *12 0 5.3 Benzene' Pttenof' 5 50 500 1000 5000 7000 5 50 500 700 1000 3000 9.45 f 1.05d 9.88 i 0.28' 10.94 i 0.41 115 8 i 0.03 10.98 f 0.37 13.22 f 0.93 10.52 f 0.17 11.08 f 0.51 13.47 f 0.95' 13.10f 0.59 16.56 f 0 34 19.50 f 0.71' 5 65 f 0 35 4 60 f 0 57 4 53 f 0 46' 4 05 i 0 21 2 95 i 1 06 2 85 i 0 64 *4 70 0 28 3 95 f 0 21 2 73 f 0 95' 2 45 i 0 07 2 05 f 0 21 0 20 It 0 00 19.5 f 0.7 18.0 i 2.8 21.o f 2.2' 27.0 f 5.7 35.5 f 3.5 35.0 i 0.0 21.0 i 1.4 16.5 f 2.1 28.0 It 8.2' 40.5 f 2.1 60.0 i 2 8 96.0 i 0.0 44.5 f 3.5 41.0 f 1.4 40.0 f 4.8 45.5 f 6.4 41.5 i 2.1 46.5 f 2.1 47.5 f 3.5 43.5 f 0.7 44.5 f 2.6 52.0 It 0.0 37.0 f 4.2 4.0 f 0.0 27.5 f 3.5 30.5 f 3.5 31.Of 4 1 22.0 i 7 1 17.0 f 4.2 16.0 f 2.8 24.0 i .I .4 29.5 i 2.1 21.0 f 7.1 7.5 f 2.1 3.0 f 1 4 8.5 f 0.7 10.5 f 5 0 8.0 f 2.8 5.5 i 4.9 6.0 f 2.8 2.5 f 0.7 7.5 k 3.5 10.5 i 0.7 6.5 rt 3.1 Catechol' I,2,4-BensenetrioQ 5 10.42 f 0.20 50 13.61 f 0 93' 70 15.12 i 0.11 4 15 f 0 07 2 18 i 0 17' 1 75 I 0 21 22.5 f 6.4 *47.0 f 6.2' 69.0 5.6 46.0 f 1 4 41.0 f 2.9 29.0 f 7.0 *26.5 f 6 4 10.0 4 7 2.0 i 1.4 5.0 f 1.4 2.0 i 1.8 100 23.00 i 0.008 0 85 I 0 07 91.o f 0.08 9.0 i 0 0 300 Cytotoxic 5 9.50 f 0.2Od 5 50 i 0 14 21.o i 2.8 46.0 i 4.2 27.0 i 2.8 6.0 f 4.2 50 13.92 i 0.32' 5 23 k 0 49' 16.5f 3.8' 41.5 f 6.6 30.0 f 4.9 12.0 f 5.4 70 14.04 f 0.79 4 30 i 0 14 19.0 f 0.0 35.5 i 7 8 30.5 f 1.4 15.0 f 2.8 100 15.52 f 0.62 3 15 f 0 35 14.0 i 1.4 47.0 f 4.9 30.0 f 1.4 9.0 f 4.2 300 22 27 f 1.27h 0 15 i 0 07 43.0 f 0.oh 46.0 f 0.0 11.0* 0.0 500 Cytotoxic Hydroquinone' 5 10.32 f 0.23 50 12.84 f 0.53' 70 12.96 i 0.45 100 15.92 i 0.17 300 Cytotoxic 4 00 f 0 28 2 30 f 0 37' 15OiO14 0 70 i 0 00 42.0 f 4.2 41 0 f 6.2' 42.5 i 2.1 58 5 f 2.1 35.5 i 2.1 41.5 f 4 9 46 5 f 2.1 36 5 i 2.1 17.5 f 4.9 13.0 i 3.6 8.0 i 4.2 5.0 i 0.0 5.0 i 1.4 4.5 i 3.1 3.0 f 0.0 1,4-~enzoquinone~ 5 12.36f 0.40 3 50 f 0 57 22.5 f 6.4 47.0 i 2.8 25.5 f 0.7 5.0 f 1.4 50 17.52 i 1.49' 70 16.74 f 0.82 *3 20 i 0 50' 2 00 0 28 19.0f 2.4' 51.Of 5.1 18.0 i 2.1 43.0 f 2.8 23.0 i 4.5 27 0 i 1.4 7.0 ~2t.4 12.0 f 6.4 100 19.08 f 0.51 195fO21 35.5 f 3.5 39 0 f 1.4 20.5 f 2.1 5.0 f 2.8 300 cytotoxic '~ . . .. .- _ _- . ~ ........ The test chemical is significantlydifferent from the concurrent control at P < 0.05, using a one-way analysis of variance for the SCE. mitotic 'index, and cell cycle kinetics data. respectively. Mean f SO among cultures. A total of 225 second-division metaphases, 9000 nuclei, and 900 coiiseciilive metaphases were analyzed for SCE, inilotic index, and cell cycle kinetics, respectively. Using a one-tailed Dunnett's multiple range test for the SCE data, 5 p~ benzene and 1,2,4,-benzenetriolare not significantly different froin the concurrent control at P < 0 05. A total of 100 seconddivision metaphases, 4000 nuclei, and 400 consecutive metaphases were analyzed for SCE, mitotic index, and cell cy$e kinetics. respectively. A total of 3 seconddivision metaphases. ''A total of 9 second-divisionmetaphasesand 50 consecutive metaphases. A total of 25 second-division metaphases and 50 ConseCLltive metaphases. CANCER RESEARCH VOL. 45 JUNE 1985 'l171 BENZENE-INDUCED SCE IN HUMAN LYMPHOCYTES derived for benzene and each known metabolite examined that showed SCEs in human T-lymphocytes from MNL cultures exposed in a significant concentration-relatedincrease in SCE frequency (45). vitro without any additional activating system. It is interesting to RESULTS note the markeddifferencesin SCE potencies of the compounds studied (Chart 2). On an induced SCE per basis, catechol Cytogenetic Analysis of Benzene and Its Known Metabolites. Benzene, phenol, catechol, 1,2,4-benzenetrioI, hydroquinone, and 1,4-benzoquinone induced significant concentrationdependent increases in the SCE frequency in human T-lymphocytes (Table 1). Based on the slopes of the linear regression curves for SCE induction, the relative potencies were as follows: catechol > 1,bbenzoquinone > hydroquinone > 1,2,4-benzenetriol > phenol > benzene (Chart 2). At the lowest concentration examined (5 p ~ ) 1,,4-benzoquinone was the most potent SCE inducer of all the compounds examined (Table 1). trans,transMuconic acid did not increase the SCE frequency significantly (Table 2). Benzene, phenol, catechol, 1,2,4-benzenetrioI,hydroquinone, and 1,4-benzoquinone caused significant depression of mitotic activity and inhibition of cell cycle progression (Table 1). trans,trans-Muconic acid did not significantly affect the mitotic activity or cell cycle progression(Table 2). Cytogenetic Analysis of the Proposed Metabolities of Benzene. 2,2'-Biphenol and 4,4'-biphenol induced a slight but statistically significant increase in SCE frequency (Table 2). However, the magnitude of the response was minimal compared to the response seen with benzene, phenol, catechol, 1,2,4-benzenetriol, hydroquinone,and 1,4-benzoquinone.1,4-Diphenoquinone did not cause a statistically significant increase in SCE (Table 2). 2,2'-Biphenol, 4,4'-biphenol, and 4,4'-diphenoquinone caused a significant reduction in mitotic activity, and both of the biphenols significantly inhibitedcell cycle progression(Table 2). was approximately 221 times more active than benzene at the highest concentrations studied. Results from the present study on SCE induction, inhibition of mitotic activity, and cell cycle progression with catechol and hydroquinone correlate well with the data of Morimoto and Wolff (36). Both studies report the following: (a) catechol is a more potent SCE inducer than is hydroquinone; ( b )catechol and hydroquinone are about equal in decreasing mitotic activity; and (c) catechol is more effective than hydroquinone in inhibiting cell cycle progression. The present study shows that 1,4-benzoquinone(theoxidized metabolite of hydroquinone) is more potent than catechol at inducing SCE at 5 and 50 PM. Ingeneral, catechol depressedthe mitotic activity and slowed the cell cycle progression more effectively than did 1,4-benzoquinone. 1,2,4-BenzenetrioI is the least cytotoxic of the 4 most reactive metabolites (catechol, 1,2,4-benzenetrioI, hydroquinone,and 1,4-benzoquinone).Thus, these 4 metabolites might be responsible for most of the in vivo genotoxicity associated with exposure to benzene. Few reports exist on the mutagenic effects of the major metabolites of benzene (phenol, catechol, and hydroquinone), and no data are available regarding the mutagenicity of its remainingknown and proposedmetabolites. Exposureto phenol, catechol, and hydroquinonein the Ames' Salmonella test with or without S-9 activationdid not increase the number of revertants (47, 48). However, Gocke et a/. (49) found that phenol with S-9 and hydroquinone without S-9 were positive in the Ames test when usingZLM mediuminstead of Vogel-Bonner medium.Also, DISCUSSION hydroquinone has been shown to induce micronuclei in mouse bone marrow (49,50)whereas catechol does not (50).Bulsiewicz These results demonstrate that benzene, phenol, catechol, (51) found a significant increase in chromatid aberrations in 1,2,4-benzenetrioI, hydroquinone, and 1,4-benzoquinoneinduce mouse spermatogonia and primary spermatocytes following p.0. exposure to phenol. Thus, mammalian systems detect the mu- tagenic effects of the metabolites of benzene, whereas the results from prokaryotic systems are equivocal. Quinone metabolites formed by the oxidation of catechol and hydroquinone are toxic to lymphoid cells and bone marrow (52- 55). Quinones and semiquinones have been implicated as the ultimate reactive metabolites of benzene in the liver (20), in human lymphocytes (37, 38), and in rabbit bone marrow nuclei (56). Irons et a/. (52) implicated 1,4-benzoquinoneas being the ultimate toxic metabolite of benzene but did not rule out the potential importance of 1,4-benzosemiquinone. They also dem- onstrated that of the metabolites studied (hydroquinone, 1,4- benzoquinone, and catechol), 1,4-benzoquinone was the most potent inhibitor of blast transformation in phytohemagglutinin- stimulated rat spleen lymphocytes. Their data on the mitogenic responsiveness after treatment with hydroquinone and 1,4-ben- zoquinone correlate well with our data on mitotic inhibition 0 200 500 I0WH 3000 5000 7000 caused by these metabolites. Irons et a/. (52) showed that 0.4 (w)CONCENTRATlON p M 1,4-benzoquinorie and 2 PM hydroquinone inhibited mitoge- Chart 2. Linear regression equations of the SCE induction curves for benzene nesis in rat splenocytes, whereas up to 10 PM catechol had no and each known metabolite that showed a significant concentration-related in- effect. In contrast with the study of Irons et a / . (52), the present crease in SCE. The best-fit linear equation and squared correlationcoefficient for each compound are as follows: (0).catechol (CT),y = 0 fZ4x .t 8.6. r z = 0.91; study shows that 5 {LM catechol in addition to hydroquinoneand +(A), 1.4-benzoquinone(BQ),y = 0 . 0 9 2 ~ 10.7,f a = 0.85; (W), hydroquinone(HO), + +y = 0 . 0 6 3 ~ 9.3, r2 = 0.95; (0).1,2,4-henzenetriol(87),y = 0.043~ 10.27. r z += 0.93; (A),phenol (PH),y = 0 . 0 0 3 2 ~ 10.9, rz = 0.84; 0 ,benzene (BZ),y = 1,4-benzoquinone inhibited mitotic activity. Hydroquinone and 1,4-benzoquinone have also been shown to be highly toxic to 0 . 0 0 0 4 ~f 9.9, r 2 = 0.62. the colony formation of mouse bone marrow stromal cells, 1 met - __ 0 3c Iran. 2,2' 4,4' 0 80 4.4' - C kinst I contt e, '1 whe pre: quir rece in tc cyte G indu mag expi 19.5 batic undi in m seer with stud nifici rnM M SCE cyte and rher CANCER RESEARCH VOL. 45 JUNE 1985 7474 BENZENE-INDUCED SCE IN HUMAN LYMPHOCYTES Table 2 n fffect of known and proposedDMSO-soluble benzene metabolites on the SCE frequency, mitotic acfivity, and cell cycle kinetics of humanperipheralblood T- O lymphocyfes exposed in vifro S The removalof blood, treatment of MNLS, culture of lymphocytes, harvest, and slide preparation were as described in "Materials and Methods." Fifly second-division ,I ietaphases. ___- - 2000 nuclei, an-d-20_0 c_on_sec-u.ti_ve _me_tap-ha_se_s w_er_e a_na_lyz_ed_for SCE, mitotic index, and cell cycle kinetics, respectively, unless noted otherwise. .__________ e Cell cycle kinetics (%) ___ Y Concentration Second Fourth __ -_e Chemical -.- ._- ---- b4 SCEs/nietaphase - Mitotic in-dex ("A) - First division _ _d-ivision - _-T- hir-d divison - -_div_isio_n_ h 33% DMSO control 9 27 f 0 48'' 4 3 0 ~ 1 3 3 ~ 17 0 14 9' 42.0 f 5.7 330*71 8.0 i 4.3 e ans,frans-Muconic acid S n 5 50 500 9 82 k 0 06 9 52 f 0 23 10 06 f 0 62 515i035 5 45 f 0 21 5 40 f 0 28 200*00 195~07 225207 44 0 f 1.7 47.0 i 0.2 44.0 f 2.8 295f07 250fOO 250+56 6.5 f 0.7 8.5 k 4.9 8.5 f 3.5 e `,2'-Biphenolc i- e E Y d ,f 11 , `S )- 5 50 70 100 300 500 0.1 0.5 5.0 7.0 10.0 50.0 9 76 f 0 62 8 90 f 0 25 10 12 f 0 45 10 06 f 0 48 10 48 f 0 06d No second division 9 46 i 0 65 '9.22 f 0.59 11.62 i 0.72d8 9.78 f 0.08 10.46f 0.519~ Cytotoxic 3 25 f 0 49 4 10 I O 21 2 50 f 0 8 5 400i028 1 95 rt 0 21 1OOkOl4 3 55 f 1 20 345f021 2 45 f 0 13' 140fOOO 2 00 i 0 00 220k28 290k85 320k155 355135 *41 O k 1 0 6 100 0 0 0 28Of71 225k07 38 5 k 6 4' 445k120 575A35 47.5 f 0.7 48.0 f 2.8 41.O f 1.4 42.5 f 3.5 47.0 f 4.9 36.0 f 4.2 31.5 f 2.1 47.0 f 2.9 40.0 i 8.5 40.5 f 3.5 245f35 190L42 21 5 f 9 2 205i07 105i49 35.5 i 12.0 42 0 f 7.1 13.0 f 6 7 15.0 f 2.8 1.5 f 0.7 6.0 f 2.1 4.0 f 1.4 5.5 f 4.9 1.5 f 0.7 1.5 f 0.7 0.5 f 0.7 4.0 f 5.7 1.5 f 1.o 0.5 f 0.7 0.5 f 0.7 b 80% DMSO control )r 10.52 f 1.47 2.75 f 0 64 190k14 40.5 f 0.7 37.5 i 2.1 3.0 f 0.0 1,4'-Diphenoquinone' 0.1 9.66 f 0.25 3.05 f 0.78 31 0 * 4 2 30.0 f 7.1 38.0f 12.7 1.o i 1.4 19 0.5 8.98 f 1.10 4.45 f 0.07 175f21 30 5 f 7.8 48.0 i 1.4 4.0 f 1.4 *tS 1.o 8.82 f 0.88 3.70 f 1.13 245f92 26.0 0.0 48.0 f 11.3 1.5 f 2.1 )I, 5.0 10.16 i 0.23 3.35 f 0.07 160i14 32.0 f 1.4 45.0 f 1.4 7.0 f 1.4 )r 50.0 70.0 11.56 f 0.51 Cvtotoxic 0.65 f 0.07 340f85 43.5 f 6.4 22.5 f 2.1 ts - . .9 Mean i SD among cultures. A total of 175 second-divisionmetaphases, 7000 nuclei, and 700 consecutive metaphases were analyzed for SCE. mitotic index, and cell cycle kinetics, respectively. st `The test chemical is significantly different from the concurrent control at P < 0.05, using a one-way analysis of variance for the SCE, mitotic index, and cell cycle 3, cinetics data, respectively. dUsinga one-tailed Dunnett's multiple range test for the SCE data, 300 PM 2,2'-biphenol and 5 and 10 MM 4,4'-biphenol are significantly different from the concurrent ;e ytrol at P < 0.05. :2 A total of 100 second-divisionmetaphases, 4000 nuclei, and 400 consecutive metaphases were analyzed for SCE, mitotic index, and cell cycle kinetics, respectively. in 'The test chemical is significantly different from the concurrent control at P < 0.05, using a one-way analysis of variance for the mitotic index data only. 0. U- le whereas catechol is considerably less toxic (57). In general, the between the Morimoto and Wolff (36) and present studies. (a) present results on the cytotoxic effects of the dihydroxy and They added benzene and phenol at culture initiation when the id !- le in lei le quinone metabolites of benzene are in agreement with other recent studies, but catechol might have a more prominent role in toxicity to human lymphocytes compared to rodent lympho- cytes. Gerner-Smidt and Friedrich (58) showed that benzene did not induce SCE in human T-lymphocytes stimulated with phytohemagglutinin. Their finding was most likely attributable to the lymphocytes were in Go-G,, whereas we added these cornpounds 24 h after mitogenic stimulation when lymphocytes would be blast transformed and in the GI-S phase. Because cytochrome P-450 (AHH)activity increases during blast transformation of human MNLs in the presence of aromatic hydrocarbons (39-41), and aryl-4-hydroxylaseis an enzyme that oxidizes benzene (59), it is probable in the present study that benzene 10 n- 4st ntic nin experimental protocol used. In their study, benzene (0.195 to 19.5 mM) was mixed with serum and injected into corked incubationflasks in which the whole blood lymphocytes were cultured under reduced oxygen tension for 72 h. A significant decrease ni mitotic activity and inhibition in cell cycle progressionwas not Seen after exposure of up to 19.5 mM. These results contrast with the findings of Morimoto and Wolff (36) and the present and phenol could be stimulating their own metabolism. Rudiger et a/. (60) demonstrated that benzo(a)pyrene induced its own metabolism in human lymphocytes as showri by an increased SCE frequency. (b) Cytogenetic lesions induced immediately prior to S phase might have little or no time to be repaired, and consequently, SCE inductionshould be greater (61).(c)Because of their high volatility, benzeneand phenolmight haveevaporated 1.4 study where aerobic-culturemethodologies were used, and sig- before DNA synthesis in the Morimoto and Wolff (36) study. (d) nificant cytotoxicity was seen with benzene concentrations of 1 Differences could exist in the SCE response between Ficoll- 18- 10 nt Id ?d to 1% mM and 50 P M , respectively. Morimoto and Wolff (36) did not observe an increase in the SCE frequency in phytohemagglutinin-stimulatedhumanlyrnphocytes from whole bloodcultures treated with up to 5 mM benzene and saw only a small increase after treatment with 1 mM phenol. There are at least 4 possible reasons for the discrepancies Paque-separatedMNLs and whole-blood cultures. Because the liver contains much higher concentrations of AHH than do hematopoietic tissues (62), use of a postmitochondrial supernatant should be an efficient method to activate benzene to genotoxic intermediates. In this regard, Morimoto (38) found that 5 mM benzene in the presence of 10% rat liver S-9 induced CANCER RESEARCH VOL. 45 JUNE 1985 4 2475 BENZENE-INDUCED SCE IN HUMAN LYMPHOCYTES 6.2 SCEslmetaphase in human lymphocytes. It is interesting to note that in the present study, 7 m M benzene induced4.5 SCEs/ metaphase without addition of any exogenous activatingsystem. Also, normal individuals can have either low (53%), intermediate (37'/0), or high (10%) AHH inducibility after exposure to polycyclic aromatic hydrocarbons (63). Thus, it is possible that benzene treatment of T-lymphocytes from additional individuals could yield results different from those reported in the Morimoto (38) and the present studies. Results obtained from the present study suggest that the metabolism of phenol to an SCE-inducing intermediate is not mediatedby myeloperoxidase. 2,2'-6iphenol, 4,4'-biphenol, and 4,4'-diphenoquinone were marginal SCE inducers compared to benzene, phenol, catechol, 1,2,4-benzenetrioI, hydroquinone, and 1,4-benzoquinone. Phenol (1 mM) induced about 8 SCEs/ cell in the present study, whereas 2,2'-biphenol, 4,4`-biphenol, or 4,4'-diphenoquinone induced about one SCE/cell at the highest concentrations that could be analyzed. Although the phenol metabolites proposed by Sawahata and Neal (29) are inducing few SCEs/cell, the biphenolic metabolites could be contributing to bone marrow cytotoxicityin vivo. The present results suggest that phenol is either being metabolized further by AHH or alone can induce SCE. In conclusion, the present study demonstrates that in addition to phenol, di- and trihydroxybenzene metabolites play important roles in SCE induction. Furthermore, the results suggest that either benzene alone can induce SCE or, a more likely possibility, that MNLs have a limited capability to activate benzene ACKNOWLEDGMENTS We thank DOiiglaS A Neptun for drawing the blood samples, James S. Bus, Byron E. Butteworth. and Richard D. Irons for reviewingthe manuscript,and Linda Smith and Joanne Quale for typing the manuscript. REFERENCES 1. Goldstein, B. D. Hematotoxicity in humans. J. Toxicol. Environ. Health. 2 (SUPPI.): 69-105, 1977. 2. InternationalAgency for Research on Cancer. IARC Monogr. Eval. Carcinog. Risk Chem. Hum., 4 (Suppl.): 56-57, 1982. 3. Rinsky, R. A,. Young, R. J., and Smith, A. B. Leukemia in benzene workers. Am. J. Ind. Med., 2: 217-245, 1981. 4. Vianna. N. J., and Polan, A. Lymphomas and occupational benzene exposure. Lancet, 7: 1394-1395.1979. 5. Vigliani. E. C. Leukemia associated with benzene exposure. Ann. NY Acad. Sci., 277: 134-151,1976. 6 Cronkite, E. P.. Bullis. J. E., Inoue. T., and Drew, R. T. Benzene inhalation produces leukemiain mlce. Toxicol. Appl. Ptiarrnacol.. 75: 358-361, 1984. 7. Maltoni. C., and Scarnato. C. First experimental demonstration of the carcin- ogenic effects of benzene. Med. Lav.. 5: 352-357, 1979. 8. Maltoni, C., Cotti. G., Valgimigli. L.. and Mandrioli. A. Zymbal gland carcinomas in rats following exposure to benzene by inhalation.Am. J. Ind. Med.. 3: 1116, 1982. 9. Maltoni. C., Conti, 8.. and Cotti. G. Benzene: A multipotential carcinogen. Resultsof long-term bioassaysperlormed at the BolognaInstitute of Oncology. Am. J. Ind. Med.. 4: 589-630, 1983. 10. National Toxicology Program. Technical Report on the Toxicology and CarcinogenesisStudies of Benzene (CAS No. 71-43-2) in F344/N Rats and B6C3F1 Mice (Gavage Studies), NTP-TR-289. Bethesda. MD: NIH. 1984. 11, Maltoni, C. Myths and facts in the history of benzene carcinogenicity. In: M. A. Mehlman (ed.). Advances in Modern Environmental Toxicology: Carcinogenicity and Toxicity of Benzene, Vol. 4, pp. 1-15. Princeton, Princeton Scientific Publishers,Inc., 1983. 12. Snyder, R.. and Kocsis, J. J. Current concepts of benzene toxicity. CRC Crit. Rev. Toxicol., 3: 265-288, 1975. 13. Infanle, P. F., and White, M. C. Benzene: epidemiologic observations on leukemias by cell type and adverse effects associatedwith low-level exposure. Environ. Health Perspect.. 52: 75-82. 1983. 14. Tough, I. M.,Smith, P. G., Court-Brown, W. M.,and Harnden, D.G. Chromo- some studies on workers exposed to atmospheric benzene. The possible influence of age. Eur. J. Cancer, 6: 49-55, 1970. 15. Goldstein, B. D., and Snyder, C. A. Benzene leukemogenesis. Environ. Sci, Res., 25: 277-289, 1982. 16. Goldstein, 8. D. Benzene is still with us. Am. J. Ind. Med.. 4: 585-5137. 1983. 17. tfaseley. D. (ed.). Inside E. P. A.. Vol. 5, No. 45, p. 13. Washington, DC: Inside Washington Publishers, 1984. 18. Gonasun. L. M.. Witmer, C.. Kocsis. J. J., and Snyder, R. Benzene metabolism in mouse liver microsomes. Toxicol. Appl. Pharmacol.,26: 398-406. 1973. 19. Tunek, A.. Platt. K. L., Bentley. P., and Oesch, F. Microsomal metabolism of benzene to species irreversibly binding to microsomal protein and effects of modificationof this metabolism. Mol. Pharmacol., 74: 920-929, 1978. 20. Tunek. A.. Platt. K. L., Przybyski, M., and Oesch. F. Multi-step metaboli activation of benzene. Effect of superoxide dismutase on covalent binding t8 microsomalmacromolecules, and identificationof glulathione conjugatesusin, high pressure liquidchromatographyand field desorption spectometry.Chern Biol. Interact.. 33: 1-17. 1980. 21. Rusch, G. M.. Leong, B. K. J., and Laskin. S. Benzene metabolism. .J. Toxico Environ. Health, 2 (Suppl.): 23-36, 1977. 22. Glatt, H. R.. Wblfel. T., and Oesch. F. Determination of epoxide hydrolasl activity in whole cells (human lymphocytes) and activation by benzoflavone: Biochem. Biophys. Res. Commun., 7 10: 525-529, 1983. 23. Teislnger. J., Bergerova-Fiserova. V., and Kudrna, J. The metabolism c benzene in man. Prac. Lek.. 4: 175-188, 1952. 24. Irons, R. D., Dent,J. G.. Baker, T. S.. and Rickert. D. E. Benzene is metabolize and covalenlly bound in bone marrow in situ. Chem.-Biot. Interact., 30: 241 245, 1980. 25. Snyder, R.. Sammett. D., Witmer. C., and Kocsis, J. J. An overview of thc problem of benzene toxicity and some recent data on the relationship o benzene metabolism to benzene loxicity. Environ. Sci. Res.. 25: 225-240 1982. 26. Lutz. W. K., and Schlatter. C. H. Mechanism of the carcinogenic action o benzene: irreversiblebinding to rat liver DNA. Chem.-Biol. Interact.. 18: 241. 245, 1977. 27. Gill, 0 . P., and Ahmed. A. E. Covalent binding of ["Clbenzene to cellula organelles and bone marrow nucleic acids. Biochem. Pharmacol., 30: 11271131, 1981. 28. Kalf. G. F.. Rushmore, T., and Snyder, R. Benzene inhibits RNA synthesis ir mitochondria from liver and bone marrow. Chem.-Riol.Interact., 42: 353-370 1982. 29. Sawahata, T., and Neal, R . A. Horseradish peroxidase-mediatedoxidation o phenol. Biochem. Biophys. Res. Commun., 709: 988-994, 1982. 30. Tice. R. R.. Costa, D. L.. and Drew, R. T. Cytogenetic effects of inhab benzene in murlne bone marrow: induction of slster chromatid exchanges chromosomal aberrations, and cellular proliferation inhibition in DBA/2 mice Proc. Nall. Acad. Sci. USA, 77: 2148-2152, 1980. 31. Tice. R. R.,Vogt. T. F.. and Costa, D. L. Cytogeneticeffects of inhaledbenzene in murine bone marrow. Environ. Sci. Res., 25: 257-275. 1982. 32. Clare, M. G., Yardley-Jones. A,, MacLean. A. C.. and Dean, 8.J. Chromosome analysis from peripheralblood lymphocytesof workers after an acute exposure to benzene. Br. J. Ind. Med., 47: 249-253, 1984. 33. Sarto. F., Cominato, I., Pinton. A. M., Brovedani. P. G., Merler, E., Peruzzi, M., Bianchi, V., and Levis, A. G. A cytogenetic study on workers exposed 10 low concentrations of benzene. Carcinogenesis (Lond.). 5: 827-832, 1984. 34. Watanabe, T.. Endo. A., Kato, Y.. Shima. S.. Watanabe, T., and Ikeda, M Cytogeneticsand cytokinetics of cultured lymphocytes from benzene-exposed workers. Int. Arch. Occup. Environ. Health, 46: 31-41. 1980. 35. Erexson, G. L.. Wilmer, J. L., and Kligerman, A. D. Inductionof sister chromala exchanges and micronuclei in male DBA/2 mice after inhalation of benzene Environ. Mulagen.. 6: 408, 1984. 36. Morimoto, K., end Wolff. S. Increase of slster chromatid exchanges and cell cycle perturbalions of cell division kinetics in human lymphocytes by benzene metabolites. Cancer Res., 40: 1189-1193, 1980. 37. Morimoto, K.. Wolff, S., and Koizumi, A. Induction of sisterchromalid ex. changes in human lymphocytes by microsomal activation of benzene metab oliles. Mutat. Res., 719: 355-360, 1983. 38. Morimoto, K. Induction of sister chromatid exchanges and cell cycle d delays in human lymphocytes by microsomal activation of benzene. Cancec Res., 43: 1330-1334,1983. 39. Bast, R . C.. Jr., Okuda. T., Plotkin, E., Tarone, R.. Rapp. H. .J.. and Gelboin. H. V. Development of an assay for aryl hydrocarbon [benzo(a)pyreneJhydrox. ylase in human peripheral blood monocytes. Cancer Res., 36: 1967-1974. 1976. 40. Busbee. D. L., Shaw, C. R.. and Cantrell. E. T. Aryl hydrocarbonhydroxylas induction in human leukocytes. Science (Wash. DC). 778: 315-316, 1972. 41. Whitlock, J. P.. Jr., Cooper, H. L.. and Getboin. H. V. Aryl hydrocarbon (benzopyrene) hydroxylase is stimulated in human lymphocytes by mitogen$ and benz(a)anthracene.Science (Wash. DC), 777: 618-619, 1972. 42. Wilmer, J. L.. Erexson, G. L.. and Kligerman, A. D. Implicationsof an elevaled sisterchromatid exchange frequency in rat lymphocytes cultured in the ab sence of erythrocytes. Mutat. Res., 709:231-248, 1983. 43. Kligerman, A. D.,Wilmer. J. L., and Erexson, G. L. Characterizationof a ral CANCER RESEARCH VOL. 45 JUNE 1985 2476 BENZENE-INDUCED SCE IN HUMAN LYMPHOCYTES lymphocyte culture system for assessing sister chromatid exchange afler In vivo exposure l o genotoxic agents. Environ. Mutagen., 3: 531-543, 1981. 44. Woltf, S., and Perry, P. Differential Giemsa staining of sister chromatids and the study of sister chromatid exchanges without autoradiography. Chromosoma (Eerl.),48: 341-353, 1974. 45. Snedecor,G. W., and Cochran, W. G. Statistical Methods, Ed. 6. Anies. Iowa: Iowa State University Press, 1967. 46. Kirk, R. E. Experimental Design: Procedures for the Behavioral Scientist, Eelmont, CA: Braoks/Cole PublishingGo.. 1968. 47. Dean, E. J. Genetic toxicology of benzene, toluene, xylenes and phenols. Mutat. Res., 47: 75-97, 1978. 48. Haworth, S., Lawlor, T.. Mortelmans, K., Speck. W., and Zeiger, E.Salmonella mutagenicity test results for 250 chemicals. Environ. Mutagen., 5 (Suppl. 1): 3-142, 1983. 49. Gccke, E., King, M -T.,Eckhardt, K.. and Wild, D. Mutagenicity of cosmetic ingredientslicensedby Europeancommunities.Mutat. Res., 90: 91-109, 1981. 50 Tunek, A., Hogstedt, B., and Olofsson, T. Mechanism of benzene toxicity. Effects of benzene and benzene metabolites on bone marrow cellularity, number of granulopoietic stem cells and frequency of micronuclei in mice. Chem.-Biol. Interact., 39: 129-138. 1982. 51 Eulsiewicz. H. The influence of phenol on chromosomes of mice (Mus miiscu/us) in the process of spermatogenesis. Folia Morphol. (Warsaw), 36; 13-22, 1977. 52 Irons, R. D.. Neptun, D A,, and Pfeifer, H. W Inhibition of lymphocyte transforniation and microtubule assembly by quinone metabolites of benzene: evidence for a common mechanism. J. Reticuloendothel. SOC.,30;359-372, 1981. 53. Pfeifer, R. W., and Irons, R. D. Inhibitionof lectin slirnulated agglutination and mitosis by hydroquinone: reactivity with intracellular sulfhydryl groups. Exp. Mol. Pathol., 35: 189-198, 1981. 54. Pleifer, R. W., and Irons, R. D. Effect of benzene metabolites on PI-IA- stimulated lymphopoiesis in rat bone marrow. J. Reticuloendothel.SOC.,31: 155-170, 1982. 55. Wierda, O., and Irons, R. D. Hydroquinoneand catechol reduce the frequency of progenitor E lymphocytes in mouse spleen and bone marrow. Immunopharmacology, 4: 41-54,1982. 56. Post, G. 8..Snyder, R.. and Kalf. G. F. Inhibitionof mRNA synthesis in rabbit bone marrow nuclei in vitro by quinone metabolites of benzene. Chem.-Biol. Interact., 50: 203-211, 1984. 57. Gaido, K., and Wierda, D. / A vitro effects 01 benzene metabolites on mouse bone marrow stromal cells. Toxicol. Appl. Pharmacol.. 76: 45-55, 1984. 58. Gerner-Smidt. P., and Friedrich, U. The mutagenic effect of benzene, toluene and xylene studied by the SCE technique. Mutat. Res., 58: 313-316, 1978. 59. Ikeda. M., Enzymatic studies on benzene intoxication. J. Eiochem. (Tokyo), 55: 231-243,1964. 60. Rudiger,ti. W.. Kohl. F..Mangels, W.. von Wichert, P.. Eartram, C. R., Wohler, W., and Passarge, E. Benzpyrene induces sister chromatid exchanges in cultured human lymphocytes. Nature (Lond.). 262: 290-292, 1976. 61. Schvarlzman, B., and Gutierrez, C. The relationship between the cell time available for repair and the effectivenessof a damaging treatment in provoking the formation of sister chromatid exchanges. Mutat. Hes.. 72: 483-489, 1980. 62. Andrews. L. S., Sasame, M A,, and Gillette, J. R . 3H-Eenzenemetabolismin rabbit bone marrow. Life Sci., 25: 567-572, 1979. 63. Kellermann, G., Luyten-Kellermann, M., and Shaw, C.R . Genetic variation of aryl hydrocarbon hydroxylase in human lymphocytes.Am. J. Hum. Genet., 25: 327-331,1973, 64. Sawahata, T., Rickert. D. E., and Greenlee, W. F. Metabolismof benzene and its metabolites in bone marrow. In: R. D. Irons (ed.). Toxicology of the Blood, pp 41-48. New York: Raven Press, 1985. CANCER RESEARCH VOL. 45 JUNE 1985 2477