Document RadaBkm3Qpqoavyq4QZ0o26Kn

,NDR. W. PFEIFER :tin- geed Lfhyd,, lectin sites 211 growth and I in Animal 183-296 (1973). release from Lfhydry1 1979). Jrmability and adenosine md izyme to 51-1569 (1978). piegesetzes n- 79:418-431 nes. -J. -her. ochalasin A in and muscle sulfhydryl 7-732 (1976). compounds views in d R. Philpot, ncipal hydroxyization. Arch. ups in polymeri54 (1974). sham and K. M. le proteins. tubulin: inding. he in vitro 226-238 (1976). ation of aracterization the GTP sites. i 1 t 7 'r'i CYTOGENETIC EFFECTS OF INHALED BENZENE IN MURINE BONE MARROW R. R. Tice, T. F. Vogt and D. L. Costa Medical Department Brookhaven National Laboratory Upton, NY 11973 INTRODUCTION Exposure to benzene is both an occupational and an environmental hazard. Health concerns are based on the extensive use of benzene in industry and local commerce (1) and a long-standing association with aplastic anemia and leukemia in occupationally-exposed workers (2-4, see Goldstein, these proceedings). Although negative for genotoxic activity in a variety of short-term in vitro bioassays (5-7), benzene is capable of inducing tumors in animals (8,9). The discrepency between these and other in vitro and in vivo results strongly suggests that a metabolite(s), rather than the parent compound, is primarily responsible for benzene's genotoxic activity. Both cellular toxicity and carcinogenic potency appear to be causally-related to an agent's clastogenic potential (10,ll). Exposure to benzene causes chromosomal aberrations in animals (1-4,121 and is associated with an increased frequency of such clastogenic events in occupationally-exposed workers (1-4,13-15). Several investigators have extended these observations in animals to other cytogenetic manifestations of DNA damage, e.g. micronuclei (16,17) and sister chromatid exchanges (SCEs) (18). Furthermore, two presumptive metabolites of benzene, catechol and hydroquinone, induce SCEs and inhibit cellular proliferation in human cells in vitro (19). Extrapolating these effects of benzene to an assessment of its impact on human health requires considerably more scientific research. Some of the needed research includes: (i) an understanding of the mechanism(s) by which the various manifestations of benzene toxicity occur, (ii) the acquisition of dose-response information by exposure routes pertinent to human experience, and (iii) an analysis of whether 258 R. R.TlCE ET AL such factors as age, sex and/or genetic constitution can modulate the magnitude of the response. Using cytogenetic methodologies based on the incorporation of 5-bromodeoxyuridine (BrdUrd) into DNA (18,20,21), we have examined benzene's ability to induce chromosomal aberrations or SCEs and to inhibit cellular proliferation in murine bone marrow. Analysis of SCE frequency appears especially useful since SCEs are, in many instances, an extremely sensitive indicator of direct DNA damage and mutagenic potential (22-25, see Galloway and Tice, these proceedings) Inhibition of cellular proliferation may be a sensitive measure of cellular toxicity. Consequently, the former endpoint may indicate leukemogenic potential, while the latter endpoint, bone marrow depression leading to anemia. We have exposed mice to benzene by inhalation, the normal route for human exposure, and have attempted to assess: (i) benzene's ability to induce different kinds of genotoxic damage, (ii) the importance of concentration, age, sex and genetic constitution in modulating benzene's genotoxic potency, and (iii) the relationship between liver-specific metabolism and extrahepatic metabolism in determining the observed cytogenetic effects. MATERIALS AND METHODS In all of these studies, mice were obtained as weanlings from Jackson Laboratory, ME, and fed and housed under the conditions described in detail elsewhere (18). Inhalation exposures were conducted in an isolation chamber previously described (26). Benzene vapor was generated by bubbling filtered compressed air through liquid benzene and diluting the vapor effluent appropriately. Benzene concentrations were measured at half-hour intervals by a gas chromatograph (Packard, Model 417, equipped with a column of 10% silicone SE-30 on Chromasorb W-HP) using an automatic sampling valve. Some animals were .injected intraperitoneally (IP) with benzene dissolved in corn oil (Mazola) With one exception, mice were infused with BrdUrd (Sigma; 50 mg/Kg per hour) as described in detail elsewhere (21) beginning one hour after completion of the benzene exposure and extending for up to 30 hours. Two hours prior to animal sacrifice, Colcemid (Gibco; 1 mg/Kg) was injected intravenously into each animal. Femoral bone marrow was obtained by flushing the femurs with phosphate buffered saline (pH 7.0), incubated in 0.075 M KC1 and then fixed with 3 : l methano1:glacial acetic acid. Flame-dried slides were prepared and differentially stained with Giemsa (Harleco) using a modification (18,27) of the technique of Goto et al. (28). Metaphase spreads were analyzed microscopically for chromosomal aberrations (in 50 first generation metaphase cells), SCEs (in 25 second generation metaphase CYTOGENETIC EFFECTS cells) and for rep3 tions completed in metaphase cells). text. RESULTS AND DISCUS Induction of Genot In our first ly 10 months of ag am- 1 pm) (18). A nificant increase induce a significa row cells of eithc marrow of male but liferative inhibit after the exposurt delayed formation action (18). To examine t' cytogenetic manif jected with pheno barbital is a we1 (29) and has been While having no e bital: (i) enhanc cells of female u induce chromosoma but more dramatic hanced benzene's marrow of male mJ aberrations were There was also nc (18). The differei response observe1 male mice (Table results demonstr to benzene. Sec ter the magnitud bone marrow supp toxic activity. male mice, with involvement of d responses. R. R. TlCE ET AL -can 'ulate the :orPoration of have examined .or SCEs and to Analysis of re, in many inDNA damage and hese proceedings). .ivemeasure of it may indicate )one marrow de- the normal route (i) benzene's ae , (ii) the imstitution in morelationship betabolism in de- ; weanlings from le conditions deSures -.ere conduc26). :nzene vapor .through liquid benBenzene concentras chromatograph ..I silicone SE-30 on Some animals were ilved in corn oil :dUrd (Sigma; 50 :21) beginning one extending for up , Colcemid (Gibco; ~ l .Femoral bone hosphate buffered n fixed with 3: 1 were prepared and g a modification taphase spreads were ons (in 50 first ;eneration metaphase CYTOGENETIC EFFECTSOF INHALED BENZENE 259 cells) and for replicative history, i.e. the number of cell generations completed in the presence of BrdUrd (in 100 randomly selected metaphase cells). Any additional treatments are described in the text. RESULTS AND DISCUSSION Induction of Genotoxic Damage in Murine Bone Marrow: 3000 ppm Benzene In our first experiment, male and female DBA/2 mice (approximately 10 months of age) were exposed to 3000 ppm benzene for 4 hours (9 am- 1 pm) (18). A 4 hour exposure to 3000 ppm benzene induced a significant increase in SCEs in bone marrow cells of both sexes, did not induce a significant increase in chromosomal aberrations in bone marrow cells of either sex, and inhibited cellular proliferation in bone marrow of male but not female mice (Table 1). Furthermore, the proliferative inhibition observed in male mice was even greater one day after the exposure period (Table 2 ) . This observation suggests the delayed formation of toxic metabolites and/or a delayed cellular reaction (18). To examine the effects of enhanced liver metabolism on these cytogenetic manifestations of benzene's genotoxicity, mice were injected with phenobarbital prior to their exposure to benzene. Phenobarbital is a well-known inducer of hepatic mixed function oxidases (29) and has been shown to alter benzene's toxicity in vivo (1-4). While having no effect on control values, pretreatment with phenobarbital: (i) enhanced benzene's ability to induce SCEs in bone marrow cells of female mice but not of male mice, (ii) enabled benzene to induce chromosomal aberrations in bone marrow cells of both sexes, but more dramatically in male mice than in female mice, and (iii) enhanced benzene's ability to inhibit cellular proliferation in bone marrow of male mice but not of female mice (Table 1). The chromosomal aberrations were all of the chromatid- and not of the chromosome-type. There was also no increase in chromosomal rearrangements of any kind (18) The difference in both the magnitude and the type of genotoxic response observed in the bone marrow of benzene-exposed male and emale mice (Table 3) are intriguing for several reasons. First, these results demonstrate a sex difference in the sensitivity of DBA/P mice to benzene. Second, the ability of phenobarbital pretreatment to alter the magnitude of different genotoxic responses observed in the bone marrow supports the involvement of metabolism in benzene's genotoxic activity. Finally, the spectrum of response in male versus female mice, with and without phenobarbital pretreatment, suggests the involvement of different metabolites of benzene in different genotoxic responses. 260 n cFc U k a 0 0 0m W 30 Fc Fc 8 al g n al C .rl Fc Ei C .rl al 8 N 8 P a al d 3.rl w 0 m U V wal VI al V d u PI 3 M u0 h V rl al d P (d I-( .. 40 . .+I +I hle um rld un nd I-d ww S? 00 +I +I ?? ahl dd nn I-a . .w w uhl 00 . .+I +I mln dhl rid U n dn dh . .w w NU NO . .+I +I o\d ON hld nn lnrn . .w w hlN dN +I +I ?? 000 l-l nn vlln ww 31 hld +I +I ?? hlhl drl nn lnln ww 9". Nd +I+I ?? om rl *u nlnln . .w w aI- mrl +I +I SY hlln Uhl nn aa . .w w hlm 00 . .+I +I \Dul 4- UU nn 00 rlrl . .w w *I- 00 . .+I +I he4 0000 nn I-a . .w w hlhl 00 . .+I +I rlhl uu *u nn aI- ww S? Od . .m+Il-+I mcv d al al 0) 2$ 2 3 23 3 g 9: 3 g U U nd Fc (d 9U d b -E d ala (d 0 uFc g V 8E? P 0 NO 8om mu $ 2 m 2d g n C W c 91 f 0 a0) w m +I d ral V \w Vrn 0 2 8 w1waw s3 (d R. R. TlCE ET AL. u0 Fc 0 d d Fac 3m 02 Va 71 m Um Fc U w0 m al h Ud d aU (d m U a 8V d a 1 u3 u rn .) a ah d n 3rl 0 uFc \ M B V0 ln al W u (d l-l d Fac u(d rl 0P Fac a $4 (d P (d 0c 58 cal a Bo 81 awFc .rl U 0m 8 Fc wal w .arl h d l-l (d 0 d Um d U u(d m U* CYTOGENETIC EFFECT! Table 2. Ave dur exP Time Period(hrIa (i) 1-31 (ii) 21-51 aBrdUrd infusion after terminatio bAverage Generati *Statistically di test at p < 0.01 Effect of Partial We decided 1 determining benzc a partial hepatec hibit bone marrol basis for this e: co-workers (30, moval of most of of benzene to in We removed age), allowed re mals (along with four hours (9 an The results instead of the e animals without I R. R. TlCE ET AL. CYTOGENETIC EFFECTS OF INHALED BENZENE 26 1 Table 2. Average generation time in bone marrow cells during the first and second day after benzene exposure (3000 ppm-4 hr) Time Period(hr)a Exposure Group Mice (10 mo old) AGTb (hr) (i) 1-31 (ii) 21-51 Control Benzene Control Benzene Male Female Male Female Male Female Male Female 11.920.6 (5) 10.920. 4 (5) 17.251.3 (6)* 11.120.6(6) 12.2t0.8(5) 11.OtO. 5(5) 29.8+1.2(6)* 11.420.5(5) aBrdUrd infusion period (30hr) beginning 1 hr (i) or 2 1 hr (ii) after termination of benzene exposure bAverage Generation Time (see Table 1) *Statistically different from the appropriate control by Students t test at p < 0.01 Effect of Partial Hepatectomy on Benzene-Induced SCEs We decided `tofurther examine the importance of metabolism in determining benzene's genotoxic activity by examining the impact of a partial hepatectomy on benzene's ability to induce SCEs and to inhibit bone marrow proliferation in male DBA/2 mice. The theoretical basis for this experiment was derived from studies by Snyder and his co-workers (30, see Snyder, these proceedings) showing that the removal of most of the liver in rats resulted in the decreased ability of benzene to interfer with red blood cell maturation. We removed ~ 6 0 %of the liver in male DBA/2 mice (10-14 wks of age), allowed recovery for seventeen hours and then exposed the animals (along with the appropriate controls) to 3000 ppm benzene for four hours (9 am-1 pm) (31). The results (Table 4) were surprising for three reasons. First, instead of the expected doubling in SCE values for benzene-exposed animals without partial hepatectomy, we observed a six-fold increase 262 R. R. TlCE ET AL. Table 3 . Effect of benzene inhalation on various cytogenetic endpoints in bone marrow of DBA/2 mice, (10 mo old) with and without phenobarbitol pretreatment Cytogenetic End Pointsa Chromosomal Inhibition of Sex Pheno-Na SCE Abberations Cellular Proliferation Male Female no Yes no Yes + + + ft - * - + aSee Table 1 for additional information - = no significant increase + = significant increase at p<O.Ol ++= another significant increase at p<O.O1 Table 4 . Effect of partial hepatectomy on benzene (3000 ppm-4 hr)induced SCE Exposure Group Partial Hepatectomya SCE Frequency Contro1 Exposed 4.2+0.3(6) 6.1?0.6(6) 26.222.5 ( 4 ) 25.621.7 (6) a-60% of the liver removed 17 hr prior to benzene exposure; male DBA/2 (10 -14 weeks of age) bMean frequency of SCE/cell?S. E. between (n) animals CYTOGENETIC EFFECTS in SCE frequency. normal in these bel expected inhibitioi benzene's ability ' The inability toxic ability appe (30, see Snyder, t experimental data, experimental diffe of exposure (subcu hepatectomy (7 hou .12000 ppm-hr) or b SCEs) The dose of 1 hours (3000 ppm) j oxidase P450 leve: liver during the ! Thus, the most re; the difference in study (30, see SIX of iron-59 into m nonproliferating, mechanism probabl other hand, repre response to DNA d (cited earlier) s damage versus inh results may be bc may indicate a 1i erythrocyte matui metabolite(s) foi ternatively, and itself, may be rf Effect of Co-Adm To determin formation of SCE amined the abili benzene metaboli proceedings), to DBA/2 male luene (32.4 m o l 3000 ppm benzenr proliferation. pressed by %40% R. R. TlCE ET AL. s cytogenetic end10 rr Id) with ntsa hibition of ular Proliferation ne (3000 ppm-4 hr)SCE Frequency 4.2+0.3(6) 6.120.6(6) 26.222.5 (4) 25.621.7 (6) exposure; male als CYTOGENETIC EFFECTS OF INHALED BENZENE 263 in SCE frequency. Second, bone marrow cell proliferation remained normal in these benzene exposed animals instead of exhibiting the expected inhibition. Finally, partial hepatectomy did not reduce benzene's ability to induce SCEs in bone marrow cells. The inability of a partial hepatectomy to reduce benzene's genotoxic ability appears to conflict with the results of Snyder et al. (30, see Snyder, these proceedings). In the absence of additional experimental data, the difference in our results may be explained by experimental differences such as animal species (rat vs mouse), route of exposure (subcutaneous vs inhalation), recovery time after partial hepatectomy (7 hours vs 17 hours), dose of benzene (880 mg/Kg vs .12000 ppm-hr) or by the endpoint examined (iron-59 incorporation vs SCEs) The dose of benzene to which the animals were exposed for four hours (3000 ppm) induced a maximal yield of SCEs and mixed function oxidase P450 levels did not increase in the remaining portion of the liver during the seventeen hour recovery time (data not presented). Thus, the most reasonable explanation for our respective results lies the difference in the biological endpoint under examination. Snyder's study (30, see Snyder, these proceedings) examined the incorporation of iron-59 into maturing red blood cells, a process occurring in a nonproliferating, differentiating cell population and induced by a mechanism probably unrelated to DNA damage. SCE formation, on the other hand, represents events occurring in proliferating cells in response to DNA damage (22-25). In view of our experimental results (cited earlier) suggesting possible metabolite specificity for DNA damage versus inhibition of cellular proliferation, our respective results may be both correct. The results of Snyder and his colleagues may indicate a liver-specific metabolite(s) responsible for delayed erythrocyte maturation while our results may indicate a genotoxic metabolite(s) formed directly from benzene in the bone marrow. Alternatively, and contrary to in vitro data on benzene (19), benzene, itself, may be responsible for the bone marrow-induced SCEs. I Effect of Co-Administered Toluene on Benzene-Induced SCEs To determine if unmetabolized benzene was responsible for the formation of SCEs in the bone marrow of exposed animals, we next examined the ability of toluene, a well-known competitive inhibitor of benzene metabolism in vitro and in vivo (32,33, see Snyder, these proceedings), to affect the yield of benzene-induced SCEs. DBA/2 male mice (10-14 weeks of age) were injected IP with toluene ( 3 2 . 4 mmoles/Kg) immediately prior to a four hour exposure to 3000 ppm benzene (31). Toluene did not induce SCEs or inhibit cell proliferation. Although the yield of benzene-induced SCEs was depressed by ~ 4 0 %in toluene pretreated animals, many of these doubly I 264 R. R. TlCE ET AL. exposed animals exhibited extreme respiratory depression. We thought it likely that the depressed breathing rate may have contributed to the reduction in SCEs and decided to circumvent this possible confounding effect by co-administering benzene and toluene by IP injection. Benzene was dissolved in corn oil and injected IP into DBA/2 male mice (10-14 weeks of age) one hour prior to the beginning of the BrdUrd infusion, a time corresponding to the end of the inhalation exposure. Using a range of benzene concentrations inducing low to maximal yields of SCEs, we assessed the ability of equi-molar concentrations of co-administered toluene to affect the induction of SCEs by benzene (Table 5 ) . At the lowest concentration of benzene (2.25 mmoles/Kg), toluene had no effect on benzene-induced SCE yields. However, with increasing concentrations of both agents, toluene inhibited benzene's genotoxic activity to an increasing extent. Maximal inhibition of benzene' ability to induce SCEs (%go%) occurred at concentrations of benzene which, in the absence of toluene, induced maximal yields of SCEs ($26 SCE/cell @ 22.5 mmoles/Kg). Presumably, at low levels of benzene and toluene, the enzymes responsible for benzene metabolism are in excess of the total number of benzene and toluene molecules present. Only at agent concentrations exceeding the total number of enzyme binding sites would toluene inhibition of benzene metabolism be expected to occur. Bone marrow cell proliferation and yields of chromosomal aberrations remained at control values throughout this experiment (data not shown). These results demonstrate an absolute requirement for the metabolic activation of benzene prior to the production of SCEs in bone marrow cells of exposed animals. Benzene Concentration-SCE Response: Modulating Effect of Sex and Genetic Constitution Since 3000 ppm benzene induced approximately a six-fold increase in SCEs in bone marrow cells of male DBA/2 mice when 10 to 14 weeks of age, we next examined the ability of benzene to induce SCEs at much lower ambient concentrations. We also examined whether sex and genetic constitution could modulate benzene's genotoxic activity ( 3 4 ) . In this series of experiments, male and female mice (10-14 weeks of age) from two isogenic strains--DBA/2 and C57B1/6--were exposed for four hours to concentrations of benzene ranging from 28 to 5000 ppm. These two mouse strains were chosen because they differ at the arylhydrocarbon hydroxylase (Am) inducible locus, a locus associated with carcinogenic sensitivity in mice (35,36) and, perhaps, in man (37) The lowest concentration of benzene that induced a significant increase in SCEs in male and female mice of both strains was 28 ppm CYTOGENETIC EFFECT! R. R. TlCE ET AL. ssion. W e thought e cc i b u t e d t o s poe,Lble conuene by I P i n j e c - I P i n t o DBAf2 e beginning of d of the inhalaions inducing low of equi-molar cone induction of t i o n of benzene induced SCE yields. nts, toluene inng e x t e n t . Max(%go%) occurred of toluene, in- .molesfKg) Pre- enzymes responsia1 number of benconcentrations .would toluene inBone marrow itions remained at . shown). lent f o r t h e metat of SCEs i n bone et of Sex and I six-fold increase !n 10 t o 14 weeks induce SCEs a t Id whether s e x and .oxic activity (34). : (10-14 weeks of 'were exposed f o r tm 28 t o 5000 ppm. .ffer at the arylICUS associated perhaps, i n man :ed a s i g n i f i c a n t : r a i n s was 28 ppm CYTOGENETIC EFFECTS OF INHALED BENZENE 265 ???? oowm mica Ill ..... OOOOd.- . . . .+I +I +I +I +I hlhlbhlW wmddmc dl-4 . l .+I d o oolnmlno muhluJhlln W 0 .. u (d d u d G d 0) Lc 0 W PI rn P I-l I 4 d8 7 23 PI g -nc nu aG %E 4u al PI 3P ah 3 20 P nn u\o wv ".9 00 . .h+I l m+I wm nnnn u\o\oln . . . .w w v w CJWWd ooohl . . . .m+I d+I +I u u+I 3-4- dhlC-4 0 W D.l H a PI Lc PI u W d 4 a (d `0 3 rl 4 0 W Lc PI a f;t .ij 9 u rn +I d O Laac w V v, r-l rl a PI 0 85 E 8d 7 7 a 0 d rl Lc 0 0 0 2U V H H G 0 V ma e 266 R. R. TlCE ET AL. (Table 6). Concentrations of benzene lower than 28 ppm were not tested. However, differential sex and strain sensitivities to ambient benzene were readily apparent over the concentration range examined. Benzene induced more SCEs in male mice than in female mice and more SCEs in DBA/2 mice than in C57B1/6 mice. SCE frequency increased linearly to 3000 ppm benzene in the DBA/2 mice and to 2000 ppm in the C57B1/6 mice. Higher concentrations, i.e. >3000 ppm for DBA/2 mice and >2000 ppm for C57B1/6 mice, resulted in a less than maximal frequency of SCEs. Because the SCE frequency saturates at high doses of benzene when the agent is injected IP, we believe that the most plausibible explanation for this phenomenon is that the high doses of benzene affected the central nervous system of the animals which resulted in a depressed breathing rate and a decrease in the uptake of benzene via the lungs. In early epidemiolical surveys an increased susceptibility to the adverse health activity of benzene was ascribed to women ( 1 - 4 ) . However, no current survey supports a sex-related differential sensitivity in humans for either benzene-induced anemia or leukemia (1-4). This may not be surprising considering the extent of the difference in SCE levels observed between the two sexes and the confounding effect of genetic constitution on SCE yields that we have observed in mice. The ability of benzene at concentrations as low as 28 ppm to induce a significant increase in SCEs in mice, regardless of the sex or genetic constitution of the animal, is especially interesting. OSHA currently considers 10 ppm benzene to be an occupationally safe exposure level for an eight hour working day (Time Weighted Average = 10 ppm with a 25 ppm ceiling) (see Goldstein, these proceedings). These levels are within the same order of magnitude as the lowest exposure level used in our experiments, an exposure level which elicited a positive genotoxic response in mice. Although these positive effects in mice cannot be directly extrapolated to human exposure conditions and to human physiology and the relevance of SCE induction to cancer remains unknown, the data does suggest that more research is needed to evaluate the cvrrent "safe" levels of benzene. Age-Response to Inhaled Benzene In evaluating the data from these various studies, one obvious and very significant observation is the age-related differential sensitivity of mice to the genotoxic effects of benzene (Table 7). "Aged" male mice, approximately 10 months of age, exhibited only a doubling in SCE frequency while "young" male mice, 3 months of age, exhibited almost a six-fold increase in SCEs. Furthermore, while exposure to benzene inhibited bone marrow cellular proliferation in the older male mice, inhibition which was enhanced one day after the exposure (Table 2), no inhibition was observed in the younger male mice examined under the same conditions (data not shown, 38). CYTOGENETIC EFFECTS av) W W Vm a g 2 al P Wd 0 d sc av d . ral zr W V m a rl P wd 0 V R.R.TlCE ET AL. ppm -e n o t tesr i t i k - to ambient In range examined. %lemice and more iency increased t o 2000 ppm i n 30 ppm f o r DBAf2 less than maximal rates at high doses ve t h a t t h e most t the high doses the animals which ase i n the uptake usceptibility to d t o women (1-4). differential seni a or leukemia extent of t h e sexes and t h e conIds that we have n t r a t i o n s as low i n mice, regardmal, is especially ene t o be an occuforking day (Time see Goldstein, ,ame 'er of mag:peri .ts, an ex'esponse i n mice. directly extrapolaiiology and t h e iwn, the d a t a does :he cvrrent "safe" idies, one obvious !d d i f f e r e n t i a l inzene (Table 7). exhibited only a , 3 months of age, cthermore, while c proliferation in i one day a f t e r t h e t h e younger male shown, 38). CYTOGENETIC EFFECTS OF INHALED BENZENE 267 $4 $4 1 9) g n G r( v) 9) rl V 5s 9) $4 W w 0 cn g 9) 5 N 3 P cy 0 g .I4 U cd rl s(d9) GV 4.dB W OW 0 Uv a 8rl Wrl Ww 9v) \o 9) rl P w(d z smn n @-I W n W n n m W n In v n 4 v In VI hl I- 9 "1 ss:m0 0 +I +I "! "1I- N 0 +I 00 +I +I 0 O +I I- U I- I- m m 00 zn VI n VI n n VI n m WvvW W n 5 VI 9 f 9 9 9 9 m0 +I 0 +I 0 +I rl +I rl +I d +I d +I rl hl hl N "0. '24U \o a0 m +m rl 5 5 z 5n n n U n n m n VI n W WW 9 9"1In \o 0 hl Y 9 '4'4 '4 "!0 +I 0 +I 0 +I d +I 000 +I +I +I \o 13mU \o m d rl rl d z s5 5n n n n In n n U n U W WW s sm ? 9 f VI 90 0 +I +I @-I 0 rl +I +I I- N rl +I N hl +I N 0 +I d 2U I- N rl +N \o I- N hl cv U 0 hl d a0 2mrl 0 + m$4 U g 2m0 hl d \ 03 hl \o d \ Eo U I0 U \ 0 rl U VI \ hl QD 0 hl d hl d d hl %\ U \o U I0 hl *I VI rl 0 0 a 74 V rl a 9U cd n G W Urn a U 3U at a UU 3nP) a 8 El U B9) cn P) nh 5 rl w 0 $4 co0 U $4 0V $4 $4 9) 9) U a 2a (d .Iwa4 8 zU a aaca +I 9) 5l-l l-l 69v) $4 $4 cy a9) U 88 $4 cn 9) cy VI cy 0 4a ::h h rl 9) l-l i5 tvu P) rl $4 W U m rl CU 1 *(0 U cd cn 268 R.R. TlCE ET AL. Table 7. Effect of age on benzene-induced bone marrow damage in DBA/2 male mice Control Benzene* Age (mo1 SCE a Frequency AGT (hr) SCE Frequencya AGT (hr) 3 4.3f0.3(4) 12.2+0.3(4) 26.222.5 (4)' 13.520.5 (4) 10 4.6?0. 2( 6) 14.2+0.3(6) 8.5+0.6 (7)' 16.6?0.4(7)' *3000 ppm for 4 hours aMean frequency of SCE/cell f standard error of the mean between (n) animals b Average Generation Time = mean cell cycle duration 5 S.E. between (n) animals; see Table 1 for additional information CStatistically different by Student's t test at p<O.Ol The biological basis or these age differences is not known. The differences may result from alterations in metabolism, distribution of metabolites or parent compound, target cell sensitivity or to levels of DNA repair. There is no appropriate epidemiological information for age-related occupational exposure of humans to benzene suggesting an age-related differential sensitivity. However, the extensive genetic heterogeneity among humans would make the identification of age-related effects difficult to discern. ! Route of Exposure In one additional analysis, SCE data obtained from the two routes of exposure to benzene--inhaled and IP injection--were compared (Table 8). SCE frequency increased in a linear fashion as a function of benzene concentration for both routes of exposure. For one manifestation of genotoxic damage--SCEs--and under our experimental conditions, an ambient exposure of 120 ppm benzene for four hours equaled the effect of an IP injection of 1.0 mmoles/Kg. Experiments to determine the relative potency of benzene by these two routes of exposure are in progress. However, based on the known pharmacokinetic data for benzene metabolism in vivo (33,391, we have tentatively concluded that a greater bone marrow genotoxic effect results from inhaled benzene than when injected IP (40). CYTOGENETIC EFFEC Table 8 . Eff in Inhalatior .Conc (PPd Control 28 2 17 489 1018 2082 3044 5276 *3000 ppm for 4 **Single IP inje volume 0.1 mL/ "Mean frequency CONCLUSION The results potential of ber lows: (i) The dif female D B A / ~mic gest that diffei induced chromosc cellular prolift (ii) The r1 dies indicate tl genotoxic damagc be the main sit1 R. R. TlCE ET AL. m e r -row snzene* (4)' 1 3 . 5 + 0 . 5 ( 4 ) (7)' 16.6+_0.4(7)' a mean between n f S.E. nformation 3.01 s is not known. abolism, distribu1 se tivity or epidcdrological .of humans to ben- ivity However, ould make the discern. from the two routes were compared (Table s a function of benJr one manifestation tal conditions, an equaled the effect to determine the reKposure are in proc data for benzene ncluded that a inhaled benzene CYTOGENETIC EFFECTS OF INHALED BENZENE 269 Table 8 . Effect of benzene exposure on bone marrow damage in male DBA/2 mice (10- 14 weeks of age) Inhalation* .Conc (PPd SCE a Frequency Ip Injection** .Conc mmoles/Kg SCE a Frequency Control 28 2 17 489 1018 2082 3044 5276 4.320.3 (6) 7.420.5 (4) 10.320. 9(4) 12.720.9(4) 16.221.4(5) 22.221.4 (6) 26.2+2.5(4) 17.120.9(4) Contro1 .22 2.25 5.63 11.2s 22.50 33.75 45.00 6.120.3(6) 9.420.8 (4) 10.920.2 (4) 17.0+0.6(6) 21.420.6 (6) 26.4+2.1(5) 36.023.3(4) 36.422.1(4) *3000 ppm for 4 hours. **Single IP injection of benzene dissolved in corn oil, total volume 0 . 1 mIJ5 gm "Mean frequency of SCE/per cell 2 S.E. between (n) animals CONCLUSION The results of our cytogenetic investigation into the genotoxic potential of benzene in murine bone marrow can be summarized as fol- lows: (i) The different responses to benzene observed in male and in female DBA/2 mice (with and without phenobarbital pretreatment), suggest that different metabolites of benzene are responsible f o r the induced chromosomal aberrations and SCEs and for the inhibition of cellular proliferation in bone marrow tissue in older mice. (ii) The results from the partial hepatectomy and toluene studies indicate that while metabolism of benzene is necessary before genotoxic damage can occur in bone marrow cells, the liver may not be the main site of production for the SCE-inducing metabolite. 270 R. R. TlCE ET AL. (iii) A four-hour exposure to ambient concentrations of benzene as low as 28 ppm induces a significant increase in bone marrow genotoxic damage as measured by SCE formation. (iv) The modification of the magnitude of the benzene-induced SCE frequency in murine bone marrow cells by age, sex and genetic constitution emphasizes the importance of considering these parameters when attempting to extrapolate animal data to human health risks. (v) The ability to directly correlate two different routes of benznene exposure for genotoxic damage should permit a better understanding of the pharmacokinetic distribution of benzene and its metabolites in animals and a better evaluation of benzene data for human health risks. (vi) Our results suggest that the pharmacokinetic, metabolic and molecular mechanisms involved in benzene's ability to cause anemia and leukemia are extremely complex. If different metabolites of benzene are responsible for the induction of chromosomal aberrations, SCEs and cellular proliferation inhibition, then a good animal model for anemia may not be a good animal model for leukemia and vice-versa. Considering benzene's occupational and environmental importance, research in this area is not only o considerable interest but also of economic and societal importance. Furthermore, an understanding of the biological processes involved in benzene's genotoxic activity may lead to a better understanding of the human health hazards associated with the many other genotoxic agents to which man is exposed. ACKNOWLEDGEMENT This research was conducted at Brookhaven National Laboratory under contract with the U.S. Department of Energy, the U.S. Environmental Protection Agency and the National Institutes of Health. Accordingly, the U . S . Government retains a nonexclusive, royalty-free licence to publish or reproduce the published form of this contribution or allow others to do so for U.S. Government purposes. We gratefully acknowledge the editorial assistance of Drs. K. Schaich and R. D. Benz. REFERENCES 1. T. J. Haley, Evaluation of the health effects of benzene, Clin. Toxicol. 11:531-548 (1977). 2. R. Snyder and J. J. Kocsis, Current concepts of chronic benzene toxicity, CRC Crit. Rev. Toxicol. 3:265-288 (1975). 3. E. C. Vigliani and A. 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Drew, Cytogenetic effects of inhaled benzene in murine bone marrow: Induction of sister chromatid exchanges, chromosomal aberrations and cellular proliferation inhibition in DBA/2 mice, Proc. Natl. Acad. --Sci. USA 77~2148-2152(1980). 19. K. Morimoto and S. Wolff, Increase of sister chromatid exchanges and perturbations of cell division kinetics in human lymp. ho- cytes by benzene metabolites, -Cancer R-es. 40:1189-1193- (1980). 20. S. A. Latt, Microfluorimetric detection of deoxyribonucleic acid replication in human metaphase chromosomes, Proc. ----Natl. Acad. Sci. USA 70:3395-3399 (1973). 21. E. L. Schneider, R. R. Tice, and D. Kram, Bromodeoxyuridine differential chromatid staining techniques: A new approach to examining sister chromatid exchange and cell replication kinetics, &, "Methods in Cell Biology", D. M. Prescott, ed., Academic Press, New York, NY 20:379-409 (1978). 22. H. Kat0 and H. Shimada, Sister chromatid exchanges induced by Mitomycin C: A new method of detecting DNA damage at the chromosomal level, Mutat. Res. 28:459-464 (1975). 23. P. Perry and H. J. Evans, Cytological detection of mutagencarcinogen exposure by sister chromatid exchanges, Nature 258:121-125 (1975). 24. T. Nakanishi and E. L. Schneider, In vivo sister chromatid exchange: A sensitive measure of DNA damage, Mutat. Res. 60:329-337 (1979). 25. A. V. Carrano, L. H. Thompson, P. A. Lindhl, and J. L. Minkler, Sister chromatid exchange as an indicator of mutagenesis, Nature 271:551-553 (1978). 26. S. Laskin, M. Kuschner, and R. T. Drew, Studies in pulmonary carcinogenesis, &, "Inhalation Carcinogenesis'', M. G. Hanna, P. Nettesheim, and J. R, Gilbert, eds., U.S.A.E.C. Symposium Series 18, Springfield, CONF-691001, CFSTI, NBS, U. S. Dept. of Commerce, 321-350 (1970). 27. R. R. Tice, M. A. Bender, J . L. Ivett, and R. T. Drew, Cytogenetic effects of inhaled ozone, Mutat. Res. 58:293-304 (1978). 28. K. Goto, T. Akamatsa, H. Shimazu, and T. Sugiyama, Simple differential. Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining, Chromosoma 53:223-230 (1975). 29. A. H. Conney, R. Welch, R. Kuntzman, R. Chang, M. Jacobson, M. Finster, and J. A. Wolff, Effect of environmental chemicals on the metabolism of drugs, carcinogens, and normal body constituents in man, Ann. N. Y. Acad. Sci. 179:155-172 (1971). 30. D. Sammett, E. W. Lee, J. J. Kocsis, and R. Snyder, Partial hepatectomy reduces both metabolism and toxicity of benzene, -J. Toxicol. Environ. Health 5:785-792 (1979). CYTOGENETIC EFFECTS 31. R. R. Tice, T, benzene in partial he] or public2 32. L. S. Andrews! R. Snyder, and hemopo: 26:293-300 33. A. Sato and T, between bel Appl. Phari 34. R. R. Tice, T , benzene in sex and gei 35. D. W. Nebert, The locr metabolize &I",Polyc: Gelboin ant NY, 346-38! 36. D. W. Nebert i tion of thc environment monooxygen; (1979). 37. B. Paigen, H. K. Paigen, hydrocarbox relationsh: and Cancer' Academic PI 38. R. R. Tice, UI 39. D. E. Rickert R. D. Iron! by inhalat: 40. D. L. Costa ai DISCUSSION Q. Ma (Western 11: 3,000 ppm benzene c chamber or as a COI A. Tice (BNL): 11 four-hour period ai Q. Sivak (Arthur 1 ppm of benzene in centrations. 1 R.R.TlCE ET AL. inetic effects of iction of sister 5 an dlular 3 . NaLi. Acad. hromatid exchanges in human lympho0:1189-1193 xyribonucleic omes, Proc. odeoxyuridine A new approach cell replication M. Prescott, ed., 78). nges induced by damage at the 1975). n of mutagenhanges, Nature er chromatid e, Mutat. Res. nd J. L. Minkler, f mutagenesis, s in pulmonary _.sis" , G. Hanna, .A.E. Symposium NBS, U. S. Dept. T. Drew, Cyto-S. 58:293-304 a m , Simple matids after sure to light 3:223-230 (1975). M. Jacobson, ronmental chemis , and normal Sci. 179~155-172 yder, Partial :icity of benzene, 1. I CYTOGENETIC EFFECTS OF INHALED BENZENE 273 31. R. R. Tice, T. F. Vogt, and J. L. Ivett; Cytogenetic effects of benzene in murine bone marrow. 11. The effect of prior partial hepatectomy or co-administered toluene, submitted for publication. 32. L. S. Andrews, E. W. Lee, C. M. Witmer, J. J. Kocsis, and R. Snyder, Effects of toluene on the metabolism, disposition, and hemopoietic toxicity of [ 3H] benzene, Biochem. Pharmacol. 26:293-300 (1977). 33. A. Sat0 and T. Nakajima, Dose-dependent metabolic interaction between benzene and toluene in vivo and in vitro, Toxicol. Appl. Pharmacol. 48:249-256 (1979). 34. R. R. Tice, T. F. Vogt, and D. L. Costa, Cytogenetic effects of benzene in murine bone marrow. 111. Modulating effect of sex and genetic constitution, submitted or publication. 35. D. W. Nebert, S. A. Atlas, T. M. Guenthner, and R. E. Kouri, The Ah locus: Genetic regulation of the enzymes which metaElize polycyclic hydrocarbons and the risk or cancer, -9in "Polycyclic Hydrocarbons and Cancer", 2nd ed., H. V. Gelboin and P. 0. P. T'so, eds., Academic Press, New York, NY, 346-389 (1978). 36. D. W. Nebert and N. M. Jensen, The locus: Genetic regula- tion of the metabolism of carcinogens, drugs, and other environmental chemicals by cytochrome P-450 mediated monooxygenases, CRC Critical Biochemistry 6:401-437 (1979). 37. B. Paigen, H. L. Gurtoo, J . Minowada, E. Ward, L. Houten, K. Paigen, A. Reilly, and R. Vincent, Genetics of aryl hydrocarbon hydroxylase in the human population and its relationship to lung cancer, &, "Polycyclic Hydrocarbons and Cancer", 2nd ed., H. V. Gelboin and P. 0. P. T'so, eds., Academic Press, New York, NY, 391-406 (1978). 38. R. R. Tice, unpublished data. 39. D. E. Rickert, T. S. Baker, J. S. Bus, C. S. Barrow, and R. D. Irons, Benzene disposition in the rat after exDosure by inhalation, Toxicol. Appl. Pharmacol. 49:417-423 11979). 40. D. L. Costa and R. R. Tice, unpublished data. DISCUSSION Q. Ma (Western Illinois University): I wasn't sure whether the 3,000 ppm benzene exposure was in one application into an enclosed chamber or as a continuous flow? A. Tice (BNL): It was a continuous flow measured throughout the four-hour period and represents an integrated dose. Q. Sivak (Arthur D. Little): Would you put a couple of thousand ppm of benzene in context with work-place and environmental concentrations. 274 R. R. TlCE ET AL. A. Tice (BNL): Historically, typical exposure levels were in excess of a thousand ppm in certain factories in Italy and in the U.S. In a Science article published a few years ago, benzene levels were measured in a garage during a normal paint stripping operation and the levels were on the order of a couple of hundred parts per million. The current OSHA standards set occupational exposure levels at 10 ppm for an eight-hour day. We observed a significant increase in SCEs after a four-hour exposure to 28 parts per million in the 10 to 14 week-old mice, an exposure level not greatly different from the current OSHA "safe" limits. Q. Drew (BNL): Along these same lines the Hematology Group at Brookhaven has been looking at stem cells viability in bone marrow and we see a reduction in the number of stem cells after nine daily exposures to 25 ppm. Q. Snyder (Thomas Jefferson): Ray, one question about the relationship between the benzene by IP and by inhalation. In recent years, one concept in the area of metabolic activation and induction of reactive intermediates is that toxicity seems to occur when you exceed detoxication mechanisms. This would suggest that the kinetics of the various pathways change as the dose increases. I wonder if at the lower end of your dose response curves the kinetics aren't quite different than what they are at the higher end? A. Tice (BNL): I also feel sure that the spectrum and ratios of various metabolites of benzene alters as a function of dose. From the shape of the dose response curve and for one end point--SCEs-I get a much greater induction of SCEs at the low end of the concentration scale than I would expect from the linear regression analysis. f might interpret that to mean that the responsible reactive intermediate occurs more frequently at low doses of benzene than at higher doses. At higher concentrations of benzene the metabolite(s1 capable of inducing chromosomal aberrations or of inhibiting cell proliferation kinetics may be more prevalent. Q. Wieland (BNL): Ray, do you have any hypothesis on what may account for the difference between sexes or between young and old animals? For example, do you think it might be a manifestation of a change in DNA repair capacity? A. Tice (BNL): DNA repair capacity is said to change as a function of age, although to what extent is still questionable. I expect that the differences are due more to changes in metabolism. There's a fair amount of data which shows that as animal age, the capacity to metabolize alters. I don't have a good answer for the difference between sexes. Hopefully, we will be able to examine the sex-dependent response by neonatal imprinting. 1 also don't know of any good benzene data in humans which suggests that there is a sexrelated difference in metabolism or, for that matter, an age-related CYTOGENETIC EFFECTS difference. Perhap not been looked at Q. Snyder (Thomas regard is that in t of emphasis on the there is no good el bility between malr Q. Maltoni (Italy: female rats are mo: effects. Zymbal G: in benzene-treated R. R.TlCE ET AL. Levt were in extaly and in the U . S . benzene levels were ping operation and red parts per mila1 exposure levels significant increase er million in the ,eatlydifferent from itology Group at tity in bone marrow Lls after nine daily 3n about the relationn. In recent years, and inducrion of o occur when you gest that the kinetics eases. I wonder if he kinetics aren't .rend? :trum and ratios of :tion of dose. From le end ooint--SCEs-tow t of the concen3ar regression ana3 responsible reactive IS of benzene than at zene the metabolite(s) r of inhibiting cell hesis on what may tween young and ht be a manifestation :o change as a function :ionable. I expect .n metabolism. There's nal age, the capacity swer or the difference examine the sex-dependon't know of any it there is a sexmatter, an age-related CYTOGENETIC EFFECTS OF INHALED BENZENE 275 difference. Perhaps, like with a lot of other things, it just has not been looked at closely enough. Bob, do you have any comment. Q. Snyder (Thomas Jefferson): The only interesting note in that regard i s that in the early benzene literature there is a good deal of emphasis on the greater susceptiblity of women than men. However, there is no good evidence now to indicate any difference of susceptibility between males and females. Q. Maltoni (Italy): There are experimental data suggesting that female rats are more susceptible than males to benzene's tumorigenic effects. Zymbal Gland carcinoma, which is the most common tumor in benzene-treated animals occurs only in females and not in males.