Document ZnK4K1wyEbRZEKDZoJpkV00v0

,. Enviroic menial Health Perspectives VOl. 82..m..81-89. 1989 Multiple Activation Pathways of Benzene Leading to Products with Varying Genotoxic Characteristics by Hansruedi Glatt," Robert Padykula,+ Glenn A. Berchtold,t Gabriele Ludewig,* Karl L. Platt,* Jochen Klein,* and Franz Oescli* Benzene and 13potential metabolites were investigated for genotoxicity in Salmonella typhhKn'Km and Vi9 Chinese hamster cells. In the presence of NADPH-fortified hepatic postmitochondrial fraction (S9 mix), benzene reverted his's. typkimurium strains. The effect was strongest in strain TAl53.5. Among the potential metabolites, only the hnns-1.2-dihydrodio1, in the presence of S9 mix. and the diol epoxides, in the presence and absence of S9 mix. proved mutagenic in this strain. The anti-diol epoxide was more potent than the syndiastereomer. Both enantiomers of the untidiastereomer showed similar activities. S9 mix did not appreciably affect the mutagenicity of the anti-diol epoxide. However, detoxification was observed when purified rat liver dihydrodiol dehydrogenase (EC 1.3.1.20) w a s used a t concentrations comparable to that present in the liver. The (1S-anti-diol epoxide was a much better substrate than the (1Rbenantiomer. as was true also for (1s)- ueraua (1R)-trans-1,2-dihydrodiol.The anti-diol epoxide reverted all six strains of S.@phkIIriKm used and induced all four genotoxic effects studied in V79 cells (sister chromatid exchange > acquisition of 6-thioguanine resistance, acquisition of ouabain resistance, micronuclei). However, other potential benzene metabolites showed genotoxic effects in V79 cells, as well: sister chromatid exchange was induced by the syn-diol epoxide, 1,2.4-trihydroxybenzene,hydroquinone, catechol. and 1,2,3-trihydroxybenzene.Elevated frequencies of micronucleated cells were observed after treatment with hydroquinone. 1,2,4-trihydroxybenzene, catechol, phenol, 1,2.3-trihydroxybenzene.and quinone. Mutations to 6-thioguanine resistance were induced by quinone, hydroquinone, 12.4-trihydroxybenzene, catechol, and the tmms-l,2-dihydrodiol. By f a r the most prominent effect in the whole study was the potent induction of gene mutations by quinone and hydroquinone. Quinone produced an approximately 100-fold increase in the frequency 6-thioguanine-resistant cells even at a concentration of 1+i. This effect was surprising, since benzoquinone induced almost no mutations in S. typhirnun'um. In the V79 cells, it did not induce mutations to ouabain resistance and sister chromatid exehange, and it induced micronuclei with moderate efficiency only. This unique and narrow spectrum of genotoxic activities differs from the broad spectrum observed with the antidiol epoxide, suggesting qualitative differences in their interaction with genetic material. 2-g 3 @ --3 3 43. L .;rtroduction Substantial evidence has accumulated on the important role mutations play in chemical carcinogenesis. This is true in particular for a number of carcinogenic polycyclic aromatic hydrocarbons (1). Epoxides, especially vicinal diol epoxides, appear to constitute their major mutagenic metabolites (1,Z). Such epoxides are carcinogenic (I), transform cells in culture (SA), and induce a wide spec- *Department of Toxicology, University of Mainz, Obere Zahlbacher jtfasse 67, D-6500 Mainz, Federal Republic of Germany. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, M A 02139. Address reprint requests to H. R. Glatt, Department of Toxicology, University of Maim, Obere Zahlbacher Strasse 67. D-6500 Mainz. Federal Republic of Germany. trum of mutations (I$$), including the reversion of a w otrophic bacteria, the acquisition of resistance toward toxic chemicals, and the activation of oncogenes. Unlike the case with polycyclic aromatic hydrocarbons, the evidence for a role for mutations is weak with the carcinogen benzene. Heritable functional changes (termed gene mutations in the present study) were usually not observed in bacteria and mammalian cells treated with benzene or benzene metabolites (6). However, chromosomal damage was detected by means of cytogenetic techniques (6-13). Interestingly, the metabolites that showed this genotoxic activity were not epoxides, but were polyhydroxylated metabolites (catechol, hydroquinone, 1,2,4-trihydroxybenzene)and quinones (9-13). These findings suggest that benzene and polycyclic aromatic hydrocarbons differ in the chemical nature as well 82 GLATT ET AL. as in the biological activities of their active metabolites. Nevertheless, the following points should prevent us from overemphasizing these differences: a) The doses of benzene required for carcinogenesisare higher by several jI orders of magnitude than those required with polycyclic aromatic hydrocarbons. Very minor metabolites and/or metabolites of relatively weak biological activity could therefore be responsible for benzene carcinogenesis; b) only very few biochemical and toxicological studies have been conducted on benzene oxides, dihydrodiols, and diol epoxides, the structural analogues of the critical metabo- lites of polycyclic aromatic hydrocarbons; and c) in short- term tests with benzene, its high volatility may pose a problem, rendering standard methods insensitive. In the past decade, we have studied intensively the mutagenicity of metabolites of polycyclic aromatic hydrocarbons and their enzymatic control (2).We in- cluded benzene metabolites sporadically in these studies. Benzene and tnzm-benzene-l,2-dihydrodioslhowed muta- genic activity in S. typhimuriumwhen a hepatic enzyme system was present. Systematic mutagenicity studies were initiated when benzene-1,2-diol3,4-oxidebsecame synthetically available (14).Knowing the clastogenic ac- tivity of polyhydroxylated metabolites, it was of partic- ular interest to examine whether multiple activation pathways exist for benzene and whether the genotoxic metabolites differ qualitatively in their effects. This required that several genotoxic effects could be determined in the same target cell system, since otherwise differences in responses could be interpreted as the result of trivial differencesbetween cells, e.g., in activation, detox- FIGUR1E. Stiuctures and metabolic relations of the investigated compounds. Full and dashed arrows indicate established and hypothetical metabolicpathways, respectively. Enantiomeric aspects are not implied in the structures. Some benzene metabolites may be formed on additional routes, e.g., the catechol possibly through further ification, or repair. Chinese hamster V79 cells were a particularly suitable e-xperimental model, since one could study at the same time the inductions of gene mutations (to ouabain and 6-thioguanine resistance), sister chro- hydroxylationof the phenol. The syw-diolepoxide is the diastereomer in which the allylic hydroxyl group and the oxirane moiety are on the same face of the benzene ring, whereas these groups are on opposite faces in the a>iti-diolepoxide. matid exchange (SCE),and micronuclei. The structures of the compounds studied are shown in Mutagenicity of benzene was observed oniy in the pres- Figure 1.All compounds,except duroquinone (which was ence, but not in the absence, of NADPH-fortified post- used for mechanistic reasons), the diol epoxides (which mitochondrial fraction (S9 mix)from rat and mouse liver have not yet been available as standards in any metabolic homogenate (Fig. 2). The most responsive strain was studies) and 1,2,3-trihydroxybenzene (an oxidation- TA1535. A %fold increase in the number of mutants sensitive isomer of 1,2,4-trihydroxybenzene)are known above control ivas observed even at a benzene concentra- metabolites of benzene, observed at least in certain in tion as low as 10ppm. However, further increases in the vitro systems. Muconaldehyde, a reactive metabolite of concentration had only a modest effect. The maximal mu- benzene that was recently detected (18,was not available tant number was about 3-fold the values for the control for this study. Also not investigated were the conjugated plates. Similar effects were then seen over a wide concen- benzene metabolites and muconic acid. tration range, with the limitation that details of the concentration-response curve may have been over- M utagenicity in Sa/monella shadowed by the unfavorable ratio of effect and variation. typhimurium For this same reason, it was impossible to detect quantitative differences in effect between the various Unlike other reports in the literature (16-20), we ob- metabolizing systems used. It would be very difficult, served a positive response of benzene in S. tgphimurium. therefore, to elucidate the activation mechanism by The major methodological difference was that in the studying the effect of modulations of the metabolizing earlier studies the conventional plate-incorporation and system. preincubation protocols were used, whereas we exposed Benzene oxide, tested only in the absence of liver S9 the bacteria to benzene vapor in desiccators. This tech- mix, did not show mutagenicity in a study conducted in nique allows much longer exposure periods than is pos- ou ' laboratory (11).-2positive result in strain TAlOO was sible for highly volatile compounds with conventional pro- reported for benzene oxide when high concentrations (z tocols. 7.1 mM) were used in a preincubation assay (22).How- MUTAGE.VXCITY OF BEAVZESEMETABOLITES 85 I,?0i 'I I 1 03 10 30 100 300 1000 BENZENE Ippm I FIGURE 2. Mutagenicity of benzene in Salmonella typkimurium TA1535 in the presence of mouse and rat liver postmitochondrialsu- pernatant (S9)preparation. Agar plates, containingbacteria (about lo8colony formingunits) and the liver preparationin an agar overk)~:, were replacedfor 18 hr in a desiccatorwith benzene. After furShu-incubationfor 2 days in the dark in the absence of benzene at C, the colonies were counted. Each type of symbol represents a adparate set of e.xperiments. the individual symbol represents the mean number of colonies from two plates. HepaticS9 fractions were prepared from male Sprague-Dawleyrats (closedsymbols)or female C57BL mice (open symbols)that had received a single IP injection of Aroclor 1254,500mgikg body weight, 6 days previously. Each incubation contained S9 hction equivalent to 50 mg (*)or 1M)mg liver (othersymbols)and the following cofactor systems: 2 m o l e NADP' and 2.5 pmole glucose &phosphate!(*), or 20 pnole NAD', 10pmole NADP-, and 5 pmole glucose &phosphate (other symbols). The oarametersof the assay not specifically mentionedwere as described ~eviously(SO). ever, the positive result essentially depended on misinterpretation of the data, involving the use of an h a p propriate method for the correction for cytotoxicity (28. Except for benzene oxide and the syn-diol epoxide, all benzene derivatives were investigated for mutagenicity in the presence and absence of rat liver S9 mix (Table 1). In TAl535, the strain showing the strongest response for bioactivated benzene, only tmm-benzene-1,2-dihydrodiol 'Fig. 3) and the diol epoxides (Fig. 4) proved positive. Mutagenicity of the Crans-1,2-dihydrodiol required the presence of a hepatic metabolizing system (Fig. 3). Cytosolic dihydrodiol dehydrogenase converts the trans-l,2-dihydrodiolto catechol (24-26), otherwise virtually nothing is known about the metabolic fate of the trans-l,2-dihydrodiol. The catechol was not mutagenic either in the presence or in the absence of liver S9 mix (Fig. 3) and, therefore, could not account for the S9 mixrnediated mutagenicity of the trans-l,2-dihydrodiol. 'foreover, the cytosol was not required for the activation f the dihydrodiol, as shown in experiments where the ,ostmitochondrial fraction was substituted by a microsomal preparation (data not shown). The lunds of reactions that may occur in such a system appear t o be very limited. In fact, the only precedent is the metabolism of dihydrodiols derived from polycyclic aromatic hydrocarbons to diol epoxides (I$). The idea that diol epoxides are the mutagenic metabolites formed from the Crans-1,2-dihydrodiolis further supported by the response spectra in the series of bacterial strains used. For the tmns-1,2-dihydrodiol and the diol epoxides (as well as for benzene) the order of responsiveness was TA1535 > TAlOO > TA98 (with the other strains the data are incomplete). Two diastereomers of the diol epox- ides a r e possible, depending on which face of t h e trans-1,2-dihydrodiol the epoxide group is introduced. In strain TA1535, bioactivated Crans-l,2-dihydrodiol,syndiol epoxide, and anti-diol epoxide induced about 12,20, and 800 revertants per m o l e of test compound. In a first approximation, 60 and 1.5% of the trans-dihydrodiol would therefore have to be converted to the syn-diol and anti-diol epoxide, respectively, if this were the only active metabolite. It is therefore reasonable to suspect that at least some anti-diol epoxide is formed. trans-Dihydrodiols and diol epoxides are chiral molecules. For the anti-diol epoxides, we synthesized the individual enantiomers separately. In all six bacterial strains used, the mutagenic potencies were virtually identical for both enantiomers (data not shown). Whereas in strain TA1535 only benzene, the transl,?-dihydrodiol, and the diol epoxides showed mutagenic effects, positive results were observed for additional benzene derivatives in other strains (Table 1). 1,2,3-Trihydroxybenzene, whose metabolic formation has not yet been observed, showed relatively potent effects in several strains..l,2,4-Trihydroxybenzene, a minor metabolite of berizene, was mutagenic in strain T4104 in the presence of liver S9 mix. With hydroquinone, quinone, and catechol, small increases, in the range of the detection limit, in the number of mutants were frequently observed. While we believe in the reality of these effects, their usefulness in t h e elucidation of the activation mechanisms of benzene may be negligible. Benzene Dihydrodiols and Diol Epoxides As Substrates of Dihydrodio1 Dehydrogenase Many metabolites of polycyclic aromatic hydrocarbons do not appear to be carcinogenic, although they are potent mutagens in S. typhimurium (2). One of the additional prerequisites for appreciable carcinogenicity is resistance to detoxification (2).(122)-as well as (1Santibenzene-diol epoxide showed similar mutagenic activity in the presence and absence of rat liver S9 mix (data notshown). However, in bacterial mutagenicity assays the S9 enzymes are present in strongly diluted form, as compared to the liver. In the experiments with the benzene metabolites, including the diol epoxides, we normally used S9 fraction equivalent to 17 mg liver in an incuba- tion volume of 620 &.In a previous mutagenicity study we showed that diol epoxides derived from polycyclic aromatic hydrocarbons may be detoxified in mutagenicity assays by purified rat liver dihydrodiol dehydrogenase (27). 84 GLATT ET AL. Table 1. Mutagenicity of benzene and potential metabolites in Salmonella typhimurium.' Dose range tested. LDmb Response' Compound Benzene Benzene oxide' Phenol Hydroquinone Quinone 2.2'-Dihydrosybiphenyl 4,4'.Dihydroxybiphenyl (~tlrnns-1,Z-Dihydrodiol Catechol (&~yn-Diol epoxide (lR)-anti-Diol epoxide (IS)-anti-Diol epoxide 1,2,?-Trihydroxybenzene 1,2,3-Trihydroxybenzene s9 - -A. + + + + ++ - -+-- - + + pglplate 3-1000 ppmd 3-1000 ppmd 20-4000 100-5000 20-5000 1.25-40 50-1000 0.123-4 10-200 20-1000 20-1000 20-1000 20-1000 10-3000 3.15-3000 50-1000 50-5000 10-315 10-320 10-320 2.5-10 1-2040 6.25-200 1.25-500 pglplate NT NT NT BO00 1800 > 40 > lo00 5 70 300 300 1100 > 1000 NT iJT 380 4000 NT NT NT > 40 EO0 > 200 400 TA9'i NT NT NT TA98 N--T -TAlOO -- TAN2 NNTT NT NT NT - - - NT NT N-T - --- +- N-T N-T -- N-T -- - -r -- - + +-* NT + -+ NT + +- +.- - - - + + + -- + + - + + -+ + ++ +-+ - 'The test compound, bacteria and NADPH-fortified postmitochondrial fraction 69)from liver homogenate of Aroclor 1254-treated rats (or buffer), were preincubated for 20 min at 37OC and then added to minimal agar plates. .After incubation for 3 days, the colonies were counted. Except for the preincubation and the amount of S9 fraction-in the present study the equivalent of 17mg liver was used per plate-details of the protocol were as described in (4). For technical reasons, 8 modified protocol was used for benzene (exposure in desiccators). For historical reasons, no preincubation was made with benzene oxide and tmns-1,2.dihydrodiol, and for the S9-mediated experiments with the latter compound, the equivalent of 50 mg liver was used. Toxicity was determined using his + mutants as an internal standard (31). c( + +)"he colony number at the optimal dose exceeded the value of the solvent controls by a factor of >20; (+) 2- to 20-fold (TA1535.TA98) or 1.5- to 10-fold(other strains), increase in colony number at optimal dose: (-1 a t all dose levels tested. the numbers of colonies were less than the 2-fold(TA1535, TAW) or 1.5fold (other strains) of the solvent control values. In some cases (*), our data suggest the existence of a weak effect below this criterion. NT, not tested. dExposure in desiccators; see Fig. 2. 'Data from a previous study (21).Bacteria and test compound were added to plates without a preincubation. Toxicity, in form of dilution of the his- background lawn, was observed at doses > 2 0 0 pg. 'Racemic anti-diol epoxide, at a dose of 1000pg per plate (which is more than that used with the individual enantiomers), led to a 2-fold increase in t h e number of mutants above the value of the controls. With the same protocol, 0.3 units of dihydrodiol dehydrogenase, which is the amount of enzyme present in 120 mg liver, reduced the mutagenicity of anti-benzenediol epoxide in S. typhimuriumTA1535 by 17%(icuba- tion volume = 620 pL);0.6 units (equivalent to 240 mg liver) led to a reduction of 35%, but a further increase in the amount of enzyme to 2.4 units (equivalent to 960 mg liver) showed no additional protective effect. The prod- ucts formed by dihydrodiol dehydrogenase from anti-diol epoxide have not yet been quantitatively identified;how- ever, formation of 1,2,4-trihydroxybenzene (a nonmutagen in strain TA1535) was observed. Kinetic analysis in biochemical assays (Table 2) shows that the racemic anti-diol and syn-diol epoxides are rela tively good substrates. Their Vmax/Km values were 5 and 18%, respectively, of the value for truns-l.2-dihydrodio1, which is a good substrate. Dihydrodiol dehydrogenase may therefore detoxify benzene-diol epoxides by two mechanisms, sequestration of the metabolic precursor and inactivation of the already formed diol epoxides. However, it is noteworthy that only one enantiomer of trans-benzene-l,2-dihydrodioalnd one enantiomer of the anti-diol epoxide [in both cases the (1s)enantiomer] are good substrates. Unfortunately, it appears that the (1R) tranS-1,2-dihydrodiolis preferentially formed in the liver (24).Nevertheless, Billings observed the formation of catechol from benzene in isolated hepatocytes via the di- hydrodiol pathway (26). Genotoxicity in Chinese Hamster V79 Cells The results from the genotoxicity e,xperiments in V79 cells are summarized in Table 3 (gene mutations to 6-thioguanine resistance), Table 4 (induction of micronuclei), and Table 5 (induction of SCE). Usually the compounds were tested up to their cytotoxic limits. (These limits varied with the end point. For the detection of gene mutations, a substantial fraction of the cell population has to survive for many generations, whereas initial growth retardation is acceptable. Long-term survival is dispensable for the cytogenetic assays, but two rounds I I I 1 I Table 2. Benzene dihydrodiols and diol epoxides as substrates of rat liver dihydrodiol dehydrogenase.' Compound Vmax/ffmm, L/min/mg protein truns-1,Z-Dihydrodiol Racemic (1STEnantiomer (1RbEnnntiomer 2.8 6.0 0.12 /t100 anti-Diol eposide Racemic (1STEnantiomer ( 1R)Enantiomer sytt-Diol eposide Racemic 0.14 0.25 0.014 0.52 ull T h e enzyme was purified to apparent homogeneity from rat liver (25). Activity was determined spectrophotometrically at 3T5C by following 6 NADPH production. The assay mixture contained N.IDP' r2.3 mM), ____________L the substrate and Tris-HC1buffer (30mM. pH 9.0). W> ~ ' - ~ - ~ - 4~..-...~....I-.~...._~~ . _~-.--~-.--0-~.- ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ a . +I 0 1 2 35 TEST COMPOUND [ mg per plate I First investigated was anti-diol epoxide, the putative principal active metabolite of benzene in the Ames test. FII;:RE 3. Mutagenicity of trans-benzene-1,2-tlihydrodio(l0.0a)nd c:::echol<., 0 )in SalmonellatyphiwuriumT.41535 in the presence LjuSed symbols)and absence (open symbols)of NADPH-fortified postmrtrhhondrial supernatant (S9) fraction from liver homogenate of #.r&lor 1254-treated male Sprague-Dawley rats. The plate- Racemic anti-diol epoxide induced SCE, mutations to 6-thioguanine resistance, as well as micronuclei. Signifi- a,cant effects were observed at concentrations of 10, 50, and 250 respectively. A fourth end point, induction illcorporation protocol was used and each plate received S9 equiva- of mutations to ouabain resistance, was studied using the lent to 100 mg liver tissue. Details of the assays were as described previously ( I ) . individual enantiomers of the anti-diol epoxide. Both enantiomers showed significant, but weak effects, as they of replication are required for the detection of SCE and at least one cell division is necessary for the formation of micronuclei. In the SCE assay, but not in the micronucleus assay, cells fulfilling these requirements can bh3y 'hliesrtilnegvueilsshoefdcfyrtoomtoxoitchietyr.)cells, permitting scoring at did with regard of 6-thioguanine resistance (data not shown). The (1R)enantiomer was slightly more active than the (1s)enantiomer in respect to 6-thioguanine resistance, whereas the reverse was true with respect to ouabain resistance. The syn-diol epoxide was studied for induction of SCE and micronuclei. Ten times higher concentrations than of the anti-diol epoxide were required for the equal effect on SCE, whereas an induction of micronuclei was not observed even a t very high concen- tration of the syn-diol epoxide. Quinone was the most cytotoxic of all benzene deriva- tives tested. Much to our surprise, it was a potent inducer of gene mutations to &thioguanine resistance. Esposure at a concentration of 1 led to a 100-foldincrease in the mutation frequency above the control value (from 4to 400 x loe6)A. t the most effective concentration (1.7 piIM)it in- duced 700 mutants per lo6cells, which is about 10 times the effect of the anti-diol epoxide a t the optimal concen- tration (400 pI.Therefore, if effect and exposure concen- tration are taken into account, the quinone was about 2000 times more potent than the anti-diol epoxide. The 0 10 315 100 315 1000 mutagenic activitiy of quinone (410 mutations/106 TEST COMPOUND [pg per platel cellslpM) was similar to those of some bay-region diol epoxides derived from polycyclic aromatic hydrocarbons. FIGURE 4. Mutagenicity of anti-benzene-diol epoxide (C)and synbenzene-diol epoxide (0)in Sulmaella typhimuriumTA1535. The test compound (dissolved in 10 pL dimethylsulfoxide:tnethylamine, lOCiH), 100 p L of the bacterial resuspension (1.04 x lo8colony forming units) and 500 pL buffer (150mi KCI, 10 mi sodium phosphate Under comparable, but not identical conditions, the antiand syn-chrysene-1,2-diol 3,4-oxides and the anti- and syn-9-hydroxychrysene-1,2-diol3,4-oxidiensduced 75, 9, 130,and 18mutations/106 cellsip&&r,espectively (A).How- buffer, pH 7.4) were incubated for 20 min a t 37oC. Soft agar was then ever, in contrast to diol epoxides derived from polycyclic added and the mixture poured onto minimal agar plates (27). After incubation for 3 days at 37OC in the dark, the colonies were counted. Values are means from two plates, the average standard deviation being 10.7% of the mean. aromatic hydrocarbons and benzene, quinone did not induce mutations to ouabain resistance. The ratio of the number of induced mutations, resistant to 6-thioguanine -1.a. 3 2 4 86 GLATT E T A L . Table 3. Induction of gene mutations (6-thioguanine resistance) in V79 cells by benzene derivatives.' in column 3. The mutation stability of ;he test compound, its distribution between cells and medium, and potential nonlinearities in the concentration-response curves. ,j& Table 4. Induction of micronuclei in Vi9 cells by benzene derivatives.' Compound OptimaYmaximal Frequency of concentration, micronucleated cells x 10' f l above backgroundb Phenol Hvdroauinone Qknonk trans-12-Di~ydrodiol Catechol anti-Diol epoxide *-Diol eposide 1,2,4-Trihydroxybenzene 1,2,3-Trihydroxybenzene Duroquinone 4000 17.5' 6.T 4000 25' 250 loo0 25 50' 50 35 88 27 3 35 21 1 52 32 30 'Induction of micronuclei was studied as described (33).Briefly, a total of 2 x lo5 ceUs and 4 mL medium were put into 6-cm Petri dishes. On the next day. the test compound was added, whereby at least 5 concentrationswere used. 24 hr later, the cells were harvested, treated with hypotonic KCI-solutionand fixed. Mer staining with Giemsa, 2000 cells were scored per data point for micronuclei. In control cultures, the '.m u e n c y of rim-nucleated cells was usually approximately10 x 10- Column 2 s h o w the concentration which gave the strongest effect or, with the inactire compounds (hans-1,Zdihydrodioland syn-diolepoxide), the highest concentration used. 'Except for tmn.s-1,2-dihydrodioIand symdiol epoxide, all values are considered ouiside accidental variation. 'Higher concentrations were used; however, the frequency of the micronucleated cells declined toward the value of the solvent controls. This phenomenon is attributed to the inhibition of cell proliferation. and ouabain. respectively, was 3 for the positive control, N-methyl-X'-nitro-N-nitrosoguanidin4e,for (lS)-anti- benzene-diol epoxide, about 20 for (lR)-anti-benzene-diol epoxide and > 100 for quinone. Furthermore, quinone did not induce a significant increase in SCE, and the frequency of micronucleated cells was increased by a factor of only three above spontaneous levels. Thus, quinone showed a much narrower spectrum of genotoxic activity in V79 cells. than did the anti-diol epoxide. Hydroquinone was similar to quinone with regard to the induction of &thioguanine resistance, except that about three rimes higher concentrations were required. %. 52 Table 5. Induction of sister chromatid exchange (SCE)in V79 celb by benzene derivatives.' - 4, Compound OptimaUmaximal Number of SCE p e r z concentration, pM above background d Phenol Hydroquinone Quinone trans-1 .Z.Dihydrodiol Catechol anti-Diol epoxide syn-Diol epoxide 1,2,4-Trihydroxybenzene 1.2.3-Tnhydroxybenzene 1000 20 8 4000 12.5 100 loo0 10 25 2.2 3.4' 22..00 5.St 15.5' 10.6' 7.3+ 3.2' f 1* 5 : r < 'Induction of SCE was studied as described (83).Into 6 cm Petri dishes were given at the times shown in parentheses: 4 mL medium and 2 x Id cells (0 hr), the test compound (24hr). 10 5-brom~2'-deoxyuidine (25 hr), and 0.2 pg/mL colcemid (47 hr). Cells were harvested (51 hr), treated with hypotonic XCI, fixed, and stained using the Hoechst 332jS/UV/Giemsaprocedure. A total of 30 metaphases was scored for SCE per data point. With all compounds, including those without a signlficant effect, the number of SCEs was highest a t the highest concentration with differential staining. The number of SCEs(mean f SD) in the control cultures of this study varied from 6.2 f 2.9 to 8.4 f 2.t. The t-test was used for statistical analysis. Statistically signiricant, p > 0.05. 'Statistically significant,p > 0.01. However, hydroquinone was substantially more effective than quinone in the induction of micronuclei. It was a bet- ter inducer of SCE as well. Catechol induced ail three genotoxic effects studied. The main difference it had from its isomer, hydroquinone, was its low efficiency in the induction of 6-thioguanine- resistant mutants. In its relative activities in the induction of gene mutations, SCE, and micronuclei, it was more similar to the anti-diol epoxide than to hydroqui- none and quinone. In addition, it arrested cells in the first mitosis (data not shown). This was the reason it could be studied only at relatively low concentrations in the SCE assay. Phenol and the trans-1,2-dihydrodiol showed very low MlITACENICITY OF BENZENE METAROLITES 87 cytotoxicity. Genotoxic effects were observed only a t benzene is both a metabolite of benzene and a product very high concentrations. Phenol was a weak inducer of formed from the anti-diol epoxide by dihydi*odiolde- micronuclei, while the tm~u-l,2-tlihytl1~otleiodl to a slight hytlivgenase. However, alternative pathways for its for- increase in the frequency of G-thioguanine-resistantcells. mation, not involving a diol epoxide intermediate, are It is not known whether these effects were caused by the conceivable. test compound itself, metabolites, or impurities. A very While it is obvious that diol epoxides are not major small amount of metabolites or impurity may be suffi- metabolites in the animal as a whole, the situation may cient, since the mutagenic activity of the tmns-1,2-tlihy- be different in an individual tissue. For example, in iso- tlrodiol and the micro~iucleus-inducingactivity of the phe- lated hepatocytes from pheiiobal.bital-treatetl rats, nol were less than 1%of the activities of the potential t~~cr~u-1,2-clihytlt~oatnlidocl atechol, which appeared to be metabolites, catechol and anti-diol epoxide, antl hydro- foimed exclusively via the tl.nns-l,2-dihydrodiol, made up quinone, respectively. 26% of the metabolites from benzene (26).This is much The minor benzene metabolites 2,2'-dihydroxybiphenyI, more than the contribution of catechol, 1,2,4-trihytlroxy 4,4'-dihydroxybiphenyl, and 1,2,4-trihydroxybenzene did benzene, and their conjugates to the recovered metabo- not show any particulai*ly marked effects. 1,2,4-Trihy- lites in animal studies. Moreover, there is no rationale for droxybenzene induced gene mutations and SCE antl the concept that carcinogenesis is evoked by a major micronuclei, t h e activities being similar t o those of metabolite. This is true in particular for benzene, which catechol. The tlihytIroxybiphenyIs were much less cyto- is less potent by many orders of magnitude than other toxic than the monocyclic compounds substituted with carcinogens. Hence, the available data on metabolism ar- two or three phenolic hydroxyl groups, and in contrast gue neither for nor against a possible role for diol epoxto these compounds they did not intluce gene mut;it'ions. itles in benzene carcinogenesis. Clarification could be 1,2,3-Trihytlroxybenzene w a s studied for induction of sought in metabolism and carcinogenicity studies with SCE and of micronuclei. In both systems, it showed posi- authentic diol epoxides and analytical studies on benzene tive responses, which however were weaker than those using authentic diol epoxide metabolites and nucleoside of its isomer 1,2,4-trihydroxybenzene. adducts as standards. Duroquinone cliffel-sfrom quinone in that it does not The diol epoxides were not the only mutagenic benzene conjug~itewith glutathione (28).However, in hepatocytes, derivatives in this study. Even more striking and impres- it oxidized glutathione, suggesting that it underwent re- hive were the high activities of quinone and hydroquinone dox cycling with formation of reactive oxygen (28). i n the mutation to G-thioguanine resistance. Quinone Duroquinone was much less cytotoxic in V79 cells th;m showctl ii niu*i*owspectrum of ge~iotoxicactivities. The \vas qiiinotie. It (lid not intluce gcnc mutations with ii (le- Iiiclc of any appi-eciableactivity i n S. h~phimtwiimtmay tection limit that was 10,009times below the activity of be attributed to toxicokinetic differences from V79 cells. quinone; however, it increased the frequency of t i i i c ~ i . o i n i c . l i ~ ; i tw~ ~l~lls. IHowevei-,such speailiitions cannot ex )lain the I;icIc of in- t I i t t * ~ i t i i~i ~ I ~ ~ ~ I I : I ~ rI t~&I I(;IiIiit-tt :IIIII S(' 4: ill ( l i t ' \'7!) (-tdls, l i L ~ ~ I S t i \ l ~tto* \t~i-1tii~~g\iiiniu~siici;illp invdvcs fiiiliiit! of synthesis, 01' defect in function, of the enzyme hypoxan- C o n c hsions thine phosphoiibosyl ti*ansfei.ase.Such pui.ely d e s t i ~ ~ c tive effects ciin be achieved by niitiiei-o~t~yspes of muta- It has been tlenionstl.ated in the present study that tions, including base substitutions, frameshifts, and large metabolites of benzene can induce heritable functional deletions. The lack of induction of ouabain resistmce changes (gene mutations) in bacterial and mammalian (usu;illy involving base substitution) and the wealiness of cells. Moi*eovei*t,he results of the bacteriiil mutagenic- the c.ytogcnctic effects p i n t to iiii tinki~owni,~nusu;ili,intl ily cspciiinc!iits provitlc circiunslantiul evidence for llic w r y selective ~nech;uiismfor the quinone mutagenicity. idea that ii diol epoxide is formed from benzene and ac- Another aspect of the mechanism involves the active counts for its mutagenicity. The anti-diol epoxide showed species. Quinones are electrophiles and may undergo ad- a broad spectrum of genotoxic activities in mammalian dition reactions with nuclcophiles. Furthermore, qui- ;it111I ~ i i d t ~ i ici ~l ~ li1l1s1c1 wits r c u i ~ t iI t~) ~tll~eLl iildic tlc11)~- iioiic's rniiy be i~ctliicctlto seini~~itinonfiew i~atliculse, g . , ilication, ;it least iis far iis the systems operative i n the through the action of one electron reductases, such as S9 mix were concerned. Partial detoxification may occur cytochrome P-450 reductase, an enzyme present in V79 in tissues with high activity of rlihytlrodiol de- cells (I. Gcmperlein, F. Oesch, antl H. R. Glatt, unpubhyytlrogenase,as is the case in rat liver. However, the (1R) lished result). The semiquinone could then t.eilct with the enantiomer of the Imns-diol epoxide is a relatively poor target molecule directly or via the formation of reactive substrate. This is the enantiomer that is expected to be oxygen species. Duroquinone cannot iiiulcrgo xltlition fwtnivl ~ ~ ~ ~ ~ ~ ~ tilii~c! ~l o~st~ci~*cioisc~lcicitivt cl~ln~ctyiil~i~lisni~~c;iclionbsu, l inay uiitletgo i.ctlox cycling i11 cells with hi*- of benzene to the (lR)-t~a~~s-1,2-dihydro(d2i4o).l mation of active oxygen (28).The lack of mutagenicity of After application of benzene to animals, a high propor- duroquinone argues therefore against a role for active tion of the dose can be recovered in the form of unmetabo- cjxygen i n the mutagenicity of quinone. Further suppoit lizetl benzene, phenol, hydroquinone, catechol, antl their lor this notion is provided in iin accompanying paper, conjugates. I t is unlikely that these metabolites are dealing with the mechanism of quinone mutagenicity (29). formed from a diol epoxide. In contrast, 1,2,4-trihydroxy- The potent mutagenicity of hydroquinone is especially 88 GLATT ET AL. noteworthy, as this compound can be directly isolated from the tissues and urine of animals treated with ben- '@G -..guardt. H., Hodgson. R. M.. Grover. P. L.,and Oesch, F. *genic and cell-transformingactivities of triol-epoxides a, zene, whereas the evidence for the formation of quinone is only indirect. Hydroquinone and its conjugates form the second major group of benzene metabolites, next to to other chrysene metabolites. Cancer Res. 4 6 4 5 5 6 ~ s 4~5, Marshall, J. C., Voudsen, K. H.. and Phillips, D. H. Activ*c-Ha-ras-1proto-oncogene by in ritm modification with a cal carcinogen, benzddpyrene diol-epoxide. Nature 310: js668~ phenol and its conjugates. Since the chemical reactivity of hydroquinone is low, one may suspect that it is metabolically activitated in the V79'cells. Identification of the iYinvolved enzyme(s) should be of the eatest interest, as hydroquinone is available in body uids and tissues of 36. (D19ea8n4.).B. J. Recent findings on the genetic toxicology OfbenzQC toluene, xylenes and phenols. Mutat. Res. 15.1: 153-181 (1-3 7. Tice, R., Costa, D.. and Drew. R. Cytogenetic effects of-' a'benzene in murine bone marrow: induction of sister chromatid& changes, chromosomal aberration. and cellular proliferation benzene-treated animals. Hydroquinone was nearly as ef- bition in DBM2 mice. Proc. Natl. .kcad. Sci. (US.)7 7 2148-2~' fective as quinone in the induction of 6-thioguanine resis- tance, but slightly higher doses were required. However, hydroquinone differed in its genotoxicity from quinone in (1980). I* 8. Siou, G., Conan, L.. and el Haitem. X. Evaluation of the &. genic action of benzene by oral administration with 2 cyto@n& techniques in mouse and Chinese hamster. Mutat. Res. 90: :$3-2i8: that it induced micronuclei with a much higher efficiency, and in that in addition it induced SCE. Therefore, it does (1981). y; 9. Morimoto. K.. and Wolff, S. Increase of sister chromatid e x c h 6 not appear likely that all the effects of quinone and all the effects of hydroquinone are induced by a single chemical and perturbations of cell division kinetics in human lymphby benzene metabolites. Cancer Res. 40: 1189-1193 (1980). ,$! 10. Tunek, A., Hogstedt, B., and Olofsson. T. Mechanism of b e m a s species. toxicity, effects of benzene and benzene metabolites on bone mni;l A further benzene metabolite for which mutagenic ac- row cellularity, number of granulopoietic stem cells and f r e q u e 4 tivity was observed is catechol. Like the hydroquinone, it induced SCE as well as micronuclei and 6-thioguanine resistance. However, its effectiveness in the induction of 6-thioguanine resistance was substantially weaker. of micronuclei in mice. Chem.-Biol. Interact. 39: 129-138 11. Morimoto, K.Induction of sister chlomatid exchanges and cen &I vision delays in human lymphocytes by microsomal activation 12. benzene. Cancer Res. 43: 1330-1334 (1983). Morimoto, K.,Wolff. S.. and Koizumi. .A. Induction of s i ste r c,w3 Several additional benzene metabolites showed geno- matid exchanges in human lymphoc>-tesby microsomal activation' _. toxic effects. These activities, however, were weak, a t least when the potential contribution of the compounds of benzene metabolites. Mutat. R e s 119: 355-360 (1983). $; 13. Erexson, G. L., Wilmer. 3. L., and Qigerman, A. D. Sister c b - ; matid exchange induction in human lymphocytes exposed to ben-, in the benzene metabolism is taken into account. We zene and its metabolites in vitro. Cancer Res. 45: ~ 4 7 1 - 2 4 " 7 ( 1 ~ , would therefore not rank them among the prime candi- 14. Aleksejczyk, R. A.,Berchtold, G. -4.. and Braun. -4. G. Benzene,. dates for the carcinogenic metabolite(s1 of benzene if genotoxicity is the essential mechanism. In this respect, preferential attention should be given to quinone, hydroquinone (with semiquinone as a potential active species), the anti-diol epoxide, catechol, and perhaps mucondialde- ocdiol epoxides. J. Am. Chem. SOC1. 07 2554-2555 (1985). 15. Latriano, L., Goldstein, B. D., ancl Witz. G. Formation muconaldehyde, an open-ring metabolite of benzene. in mouse liver microsomes: an additional pathway for toxic metabolites. Proe.; Natl. Acad. Sci. (US.)83: B56-8360 (1986). 16. Lebowitz, H.,Brusick, D., Matheson, D.. Jagannath. D. R., Reed. K': hyde (151, which was not included in this study. Consider. ing the qualitative differences in genotoxicity and postulating the requirement of several distinct mutations, Goode, S., and Roy, G. Commonly w e d fuels and solvents evalu- ated in a battery of short-term bioasays. Environ. Mutagen. 1: 172-173 (1979). 17. 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