Document 9ZQ13n7MymDO3byjRKJEjeRD

AMProc. Nad.A d scl: USA VoL 77.No.4. pp. 214845% 1980 Genetics Cytogenetic effects of inhaled benzene in murine bone marrow: Induction of sister chromatid exchanges, chromosomal aberrations, and cellular proliferation inhibition in DBA/2 mice (genetic toxicology/pollutants/clastogenic) RAYMOND R. TICE, DANIEL L. COSTA,AND ROBERT T.DREW M e d i d Department, Bmkhaven National Laboratory.Upton. New Yort 11973 Communicated by Richard B . S e t h , December 26,1979 ABSTRACT Exposure of adult male and female DBA 2 Imice to 3100 ppm benzene for 4 hr significantly increased t e frequency of sister chromatid exchanges in bone marrow cells of both sexes, inhibited marrow cellular proliferation (but only in male mice), and did not significantly increase the frequency of chromosomal aberrations in either sex. Phenobarbital pre- treatment synergistically interacted with benzene exposure to further increase sister chromatid exchanges in female mice, ainduce eater inhibition of cellular proliferation in male mice, and in uce aberrations ainsbigonthifisceaxnest.lDevuerlionfgcthhreosmecaotindd-t(ra!yeacfhtreormexopsoosmurael to benzene there was increased inhibition of cellular prolifer- ation in male mice and both new DNA damage and persistence of OMDNA damage in female mice. The differences in both the type and magnitude of the res nse of bone marrow cellular populations, as determined by E f e r e n t cytogenetic end points in male and female DBA/2 mice exposed to benzene or to phenobarbital and benzene, suggest not only that a metabolite of benzene is responsible for the observed effects, but that dif- ferent metabolites may be involved in different end points. The extensive use of benzene in industry and local commerce has resulted in both a large degree of human exposure and a considerable potential for environmental contamination (1). Acute exposure to benzene produces a dose-dependent depression of the central nervous system, whereas chronic exposure can lead to pancytopenia or aplastic anemia and, in some cases, leukemia (1-4).Both cellular toxicity and carcinogenic potential have been suggested to be causally related to an agent's clastogenic activity (5-7). Therefore, these chronic effects may be due to the observed ability of benzene to cause chromosomal aberrations in bone marrow cells of occupationally exposed workers and experimentally exposed animals (1-4). Recently, cytogenetic techniques have been developed that permit the simultaneous assessment of both sister chromatid exchanges (SCE)and cellular proliferation kinetics in vitro or in oioo (8,9). SCE induction has been suggested to correlate with an agent's mutagenic/carcinogenic potential (10-12), and perturbations in cellular proliferation kinetics should correlate with cytotoxic effects. Therefore, we examined the ability of benzene to induce SCE or chromosomal aberrations (or both) and to affect cellular proliferation kinetics in murine bone marrow. Recent evidence suggeststhat a liver-mediated metabolite(s) of benzene, rather than benzene itself, may be primarily responsible for benzene's interference with erythrocyte and leukocyte production in the bone marrow (13, 14). Phenobarbital, a well-known inducer of liver metabolism (IS), has been used to assess the effect of increasing hepatic xenobiotic metabolism on the health effects of benzene (1-4). The results of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adoertisement" in accordance with 18 U. S. C. $1734solely to indicate this fact. these studies have been largely contradictory: phenobarbita has been shown to ameliorate benzene's effects in some case and to increase it in others (1-4). To further examine this poS sible relationship between increased hepatic metabolism an( the health effects of benzene, we incorporated phenobarbita pretreatment into our experimental design. This approach, based on the simultaneousasskssment of thra separate cytogenetic end points, should allow insight into th, nature of the benzene-induced cellular events leading tobon, marrow depression and leukemia. The use of inhalation expo sure provides a system directly comparable to normal huma, experience with benzene. MATERIALS A N D METHODS Eight- to ten-month-old male (30-35g) and female (25-30 2 DBA/2 mice, obtained as weanlings from Jackson Laboratorwere used throughout the experiments. Virgin animals wer, kept at a population density no greater than one animal per 9' cm2on corn cob bedding (BedO'Cobs, J. R.Nielsen and Son. S. Windsor, CT), which was changed weekly. Food (Purin. Rodent Laboratory Chow 5001) and water were provided a( libitum. Animals were maintained on a 12-hr 1ight:dark cycl, (7 to 7) at E 0 C ;relative humidity was held at SO%, with `20ai exchanges per hr. Some animals were pretreated with sodiun phenobarbital (50 mg/kg) or with control saline injected in traperitoneally twice daily for 3 days prior to benzene exposure Randomized groups of mice were exposed in five separate run to benzene vapor (mean value = 3130 f 170 ppm) for 4 h (12,500 f 680 ppm-hr),beginning at 0900hr. Exposures wer conducted in an isolation chamber that has been describe1 elsewhere (16). Control animals were similarly treated, bu without exposure to benzene. Benzene vapor was generated b bubbling filtered compressed air throiigh iiquid benzene a i diluting the vapor effluent appropriately. Be~...1.7eneconc tration was measured at half-hour intervals by a gas chroiri.: tograph (Packard, model 417, equipped with a cohmn of 10, silicone SE-30 on chromasorb W-HP), with an automati sampling valve. One hour after exposure to benzene, exposed and contrc mice were infused with bromodeoxyuridine (BrdUrd. 51 mg/kg per hr) for various time intervals (generally, 21 hr) described (17). Two hours prior to the termination of th infusion, Colcemid (GIBCO) was injected intravenously at concentration of 1 pg/lO g body weight. Animals were killel by cervical dislocation, femurs were removed, and the marro\ was flushed out with phosphate-buffered saline (pH 7.2).Th resulting material wils treated with hypotonic KCl and fixativt flame-dried slides were prepared and stained with the Hoech: 33258 (American Hoechst)/black light/Giemsa (Harlea Abbreviation: SCE, sister chromatid exchanges. 2148 Genetics: Tice et al. - _. - Proc. Natl. h a d : Sci. USA 77 (1980) 2149 technique as described (17).For determination of chromosomal aberration frequencies, 50 metaphase cells (first generationafter benzene exposure only) were examined per animal on ran- d x e d slidesby previously described criteria (17). Five ani,.n were examined for each treatment. Where necessary, BrdUrd infusions of short duration (10or 18 hr) were run to obtain comparable proportions of first-generation to subsequent-generation metaphase cells in bone marrow preparations in all animals.For SCE studies,25 second-generationmetaphase cells were examined in each animal to determine the mean number of SCE per cell. In addition, in some animals 25 third-generation metaphase cells were examined for the number of SCE that had been induced in the first two replication cycles or in the third replication cycle. In these metaphase cells, SCE induced during the first two replication cycles appear as nonsymmetrical disruptions in the longitudinal staining pattern of chromosomes, whereas SCE induced in the third replication cycle appear as symmetricalexchanges in the longitudinal staining pattern (18).Statistically significant differences between groups of animals were determined by Stu- dent's t test at the P < 0.01level of significance. Cellular proliferaticm kinetics were din each animal by determining, in 100 metaphase cells, the proportion of cells that had replicated for one (I),two (II), or three (111) cycles in the presence of BrdUrd (19).Statistically significant differences between groups of animals were determined by xz analysis with P < 0.01 level for significance. RESULTS Inhalation of benzene by DBA/2 mice induced a significant increase (P < 0.01) in the level of SCE in bone marrow cells compared to unexposed controls (Table 1). The magnitude of t' -esponse was not significantly different between males and Ales (89%and 8296, respectively). This increase in SCE frequency occurred at a level of benzene exposure that did not significantly increase the frequency of chromosomal aberrations (Table 1). suggesting that, as for many other DNA-damaging agents (10-12), SCE induction may be a more sensitive indicator of agent activity than aberration yields. Phenobarbital pretreatment in itself had no effect on SCE or chromosomal aberration frequency. However, phenobarbital pretreatment did synergistically interact with benzene exposure to further increase the frequency of SCE and to elicit a significant level of Chromatid-typeaberrationsin bone marrow cellsof exposed animals (Table 1).This interaction between phenobarbital and benzene exhibited obvious sex differences with regard to both the type of the increased response and the magnitude of that response. Pretreatment with phenobarbital caused a further increase in SCE frequency in female but not in male mice ex- posed to benzene, and whereas chromosomal aberration yields were dramatically increased in both sexes, male mice had almost twice as many damaged cells as did females similarly treated with both agents. Although the magnitude of the increase in SCE due to ben- zene exposure alone exhibited no sex dependency, inhalation of benzene significantly retarded proliferation of bone marrow cells in male but not in female DBA/P mice (Fig. 1). This reduced replicative ability in male mice exposed to benzene is demonstrable by a significant shift in the proportions of first (I)-, second (II)-, and third (111)-generation metaphase cells observed in control animals toward a greater number of first- generation metaphase cells and fewer second- and third-gen- eration metaphase cells. Furthermore, phenobarbital, which enhanced benzene's clastogenic activity in a sex-dependent fashion, also dramatically increased the inhibitory effect of benzene on cellular proliferation in a similar manner. Phenobarbital, by itself, caused a slight alteration in the rate of cellular proliferation in both male and female mice (Fig. 1).However, after benzene inhalation, phenobarbital-pretreated male mice exhibited almost a complete retardation of proliferative capacity. No cell had divided more than once in the 25 hr after benzene exposure, compared with a normal proliferative time for bone marrow cells under these conditions of approximately 10hr per cell division (20).In female mice, the combination of benzene with phenobarbital pretreatment had no effect on the proliferative capacity of bone marrow cells. Another set of experiments, designed to examine the induction of SCE at later times after exposure (i.e., 20-30 hr and 40-50 hr), again underscores the sex-dependent effect of benzene on bone marrow proliferative capacity (Fig 2). With a W h r BrdUrd infusion, mice were infused either immediately after exposure to benzene or after approximately a l d a y delay. At both times, benzene-exposed male mice exhibited a significant inhibition of cellular proliferation when compared to control male or female mice or to exposed female mice (Fig. 2). However, the inhibition was much greater after the I-day delay than that observed for the same period immediately after inhalation of benzene. In the first 30hr after benzene inhalation, 78%of the metaphase cells had divided at least twice, whereas in an identical 30-hr period but after a 1-day delay only 1%of Table 1. Effect of inhaled benzene on SCE frequency and chromosomal aberration yields in bone marrow cells of DBA/2 mice with and without phenobarbital pretreatment Exposure SCE Chromahd aberrations' group Sex frequency n' Achromatic lesions Deletions Is0 deletions Abnormal cells. Control . M 4.6 f 0.2 6 0.068 i 0.012 F 4.5 f 0.3 6 0.092 f 0.020 *0.024 f 0.012 0.020 0.012 *0.004 f 0.004 0.004 0.004 8.8 i 1.2 10.8 f 2.2 Benzene Phenobarbital* M F M F 8.7 f0.4' 8.2 i 0.7, 4.1 f 0.2 4.2 f 0.2 io 11 I I 6 0.092 f 0.016 0.100 f 0.017 0.088f 0.018 0.080f 0.014 0.046 f 0.004 0.044 0.008 0.028 f 0.014 0.020f 0.006 0.002 f 0.002 O.Oo0 f o.Oo0 0.002 f 0.002 0.002 f 0.002 12.8 f 2.0 12.0 f L3 *10.8 2.0 9.6 f 1.2 Phenobarbitalt M 8.3 f 0.4 6 0.296 f 0.038' + benzene F 127 f 1.0, 7 0.216 f 0.0236 0.192 i 0.0% 0.096 f 0.0124 0.014 f 0.0b16 0.012 f o.m* mn frequency per cell per animal f SEM between animals is shown. -umber of animals examined for SCE data. Dab were derived from 5 animals in each group. 50 cells in each animal. t Animals were injected with sodium phenobarbital (50 mghg) twice daily for 3 days prior to benzene exposum. 4 Statisticallysignificant increaseabove appropriate'controlvalues at the 1%IeveL 42.4 & 3.6' 25.6 f 1.74 -, - I TT T -Y I II 111 I II 111 II 111 I II 111 I II 111 I II 111 I II I e- Benzene Pheno Pheno + benzene Control Benzene Pheno Pheno + benzene FIG. 1. Effect of inhaled benzene on cellular oroliferation kinetics of bone marrow cells ofDBA/2 mice. ith and without ohenobarbital (Pheno) pretreatment. The histograms present the replicative profile-that is, the relative proportions of metaphase cells that had divided for one (1). two ( I h or three (111) generations during the BrdUrd infusion period. Range bars indicate SEM between animals. The number of animals in each treatment ranged from 5 to 12. the metaphase cells had proliferated to this extent. These results support the observations of Leeet af. (21),which, based on the level of iron-59 incorporation into developing erythrocytes as a measure of benzene-induced bone marrow depression, also indicated increased depressionof bone marrow activity during the second day after benzene exposure. In an attempt to assess both the persistence and time-dependent formation of the benzene-induced lesions capable of eliciting an SCE response,we examined both the number and origin of SCE in metaphase cells that had replicated three times during a 3Ghr infusionof BrdUrd in female mice killed 31 and 51 hr after benzene inhalation. Only female mice were used because their bone marrow proliferative activity remained normal after the exposure to benzene. Using these infusion durations and time periods we were able to compare the number of SCE induced during the first two BrdUrd replication cycles (a1-21 and 21-11 hr) with those induced during the third BrdUrd replication cycles (= 21-31 and = 41-51 hr) in bone marrow cells after benzene exposure. During each successive cell generation there exist two possible mechanisms for an increase in SCE above control levels: from new DNA lesions induced during that cell cycle or from DNA lesions induced during previous generationsthat had persisted as a consequence of a lack of repair. Determination of which mechanism is involved is based on a comparison of the number of SCE formed during the first two generations of BrdUrd infusion with those formed during the third generation (18,22,23). If all of the DNA ksionsare induced prior to the first DNA synthetic period and no repair of this DNA damage takes place during subsequent replicative cycles, than the ratio of SCE formed during the first two cycles of BrdUrd incorporation to those SCE formed during the third cycle of BrdUrd incorporation will be 6:1,discounting background SCE. This ratio is based on the restriction of DNA damage to old DNA strands(i.e., strandsfree of BrdUrd substitution) and a subsequent dilution of these strands to daughter cells in a random fashion. If repair d these lesionsdoes take place after their induction of SCE in the first BrdUrd incorporation cycle, preventing subsequent f o w t i o n of SCE, then this ratio will become greatepthan 6:l;the magnitude of the increase depends directly on the efficiency of the DNA repair system(s)involved. Conversely, if new lesibs are induced after the first BrdUrd incorporation cycle, them the ratio of first-generation plus second-generation SCE to thirdgeneration SCE becomes less than 6:l.A combination of both new DNA lesions and persistence of old DNA lesions will also keep this ratio below 61. Our results (Table 21 suggest that both new lesions and persistence of old lesions occurred during the five generations of bone marrow cells examined in female mice after benzene exposure. SCE levels are still elevated above control values and, after background values are discounted, the ratios of first-generation plus second-generation SCE to thirdgeneration SCE at both 31 and 51 hr after benzene exposure (2.0:1 and 1.4:1,respectively) are significantly lower than theoretical ratios derived for SCE occurring either as a result of persistent or of new lesions alone. DISCUSSION Although benzene is clearly both clastogenic and cytotoxic in thisin oioo system, the relationship between the induced DNA damage and cellular inhibition remains unclear. First, both male and female DBA/2 mice exhibited increased numbers of SCE after exposure to benzene. Considering the suspected etiology of SCE induction, that of a replicative bypass rnechanism permitting the progression of DNA synthesis in the pres- Genetics: Tjceet 01. '"OrMale -90 -T 80 ro- - -60 c 02 50n0 -40 30 - 20- \ 10- Proc. Natl. Acad. Sci. USA 77 (1980) 2151 Female T t T T Cell 0- generation Control Time after exposure to BrdUrd. hr -1-31- I1 111 Benzene Control *-I I II 111 Benzene -21-51- I II Ill Control -I II 111 Benzene C--- 1-31 I I1 111 Control -1-51- I II 111 Benzene hG. 2. Cellular proliferation kinetics in DBAR mice infused with BrdUrd either directly after benzene inhalation or after a 1-daydelay. TL .histograms present the replicativeprofile-that is, the relative proportions of metaphase cellsthat had divided for one (I). two (II),or three ienerations during the BrdUrd infusion period. Range bars indicate SEM between animals. The number of animals in each treatment wadfive or six. ence of certain types and locations of lesions (24, !E),this in- creasepresumably reflects tbe presence of an agent capable of damagmg DNA to an equal extent in both sexes However. only male mice exhibited inhibition of cellular proliferation and this inhibition occurred in the absence of a significant increase in chromosomalaberrations. Second, phenobarbital pretreatment clearly enhanced the induction of %E, the induction of chro- mqomalaberrations,and theinhibition of cellularproliferation, Table 2. Frequencies of symmetrical and asymmetrical SCE in third-generation metaphase cells of female DBA/2 mice examined 31and 51hr after benzene exposure Time after benzene SCE' exposure. ExpMlre No.of Aaym- Symmet- Ratio hr. .- . ~ O U D animals metrical r i d A/St 31 E x p a d 6 9.1 f 0.3 3.4 f 0.2 2.&1 Control 5 4.3 f 0.3 1.0 f 0.2 51 E.possd 4 6.6 f0.2 2.8 fO.l 1.41 Contml 5 45 &0.3 1.3 f0.2 All animah were iofuredwith BrdUd for 30 hr prior to sacrifice. *Mean d u o forSCE per cell per animal SEM between animah. h n t y - f i v e third-generationmdaphasacelb were scored in each M i dfor the numbsrof W E induedin the fust two generations 3rdUrd e x p u n (asymmetrical-appearingexchanges) and for number of SCE induced in the third generation of BrdUrd ex- iiimure (aymmetrical-appeeringexchanges). Mean ratio d uymmetrid SCE to symmetrical SCE after aub- tractionofbackground ~ suggesting that a metabolitds), rather than benzene itself, was primarily responsible for these cytogenetic effects. However, because the various known metabolites of benzene were not actually measured in these experiments, it is not possible to definitely conclude whether phenobarbitol pretreatment al- tered the peak levels of certain metabolites by increasing the rate of benzene metabolism, altered the metabolic profile by inducing new pathways for benzene metabolism, or, in fact, interacted with benzene in a manner completely independent of phenobarbitol's known ability to enhance hepatic m e t a b lism. The sex-related differences in both the type and magni- tude of the observed responses suggest that phenobarbital is acting to alter benzene's metabolism and that different me- tabolites may have been responsible for the increases in these different cytogenetic endpoints. Although sex differences in the response of animals to benzene have also beem reported for rats (26). there isno real evidence to suggest that femalehumans react differently from male humans when exposed to benzene (1-4. We cannot conclude whether the proliferative inhibition observed in male mice exposed to benzene is due to an elon- gation of thecell cycle or to a reduced ability forcellsto enter new generations The restriction of chromosomal aberrations to chromatid type,as observed in this study and in an earlier study (27).is consistent with the type of damage induced by inhibiton of DNA synthesis (28). &cause these agentsalso re- tard cell proliferation, our results may indicate a similar kind of interaction during the 2 days after benzene expuswe in male mice.Yet female mice also exhibited an increase in this type of chromosomal aberration, but with no apparent decline in . .-1.2 - .I :.wI * >. _& bone marrow proliferativecapacity. Theinaease in inhibition obeenad in male mice aftez a l d a y delay between benzene exposure and BrdUrd infusion may indicate either an increase in thelevel of a metabolite(s)of benzene capableof inhibiting 7. Dewey, W.C,Miller.H. H.& bqm,D.B.(1971) A d . sd.USA 68,667-67 1. 8. khneider. E. L,Tie, R. & Kmm, D. (1978)in M d Bbbgv.ed. Racott, D.M.(Aadcmlc,New Y d g ' cellular palieration or a delayed response to an earlier dam- LGed* g eve+). Previous studies based on the use of Mtiumbenzene have concluded that metabolism and DNA 9. binding in mice is essentially completewiwithin l@hr after benzene exposure (29). This observation suggests t h t the in- 10. creased inhibition may r d from a delayed response. Thisstudy suggests that the relationship between exponue to benzeneand ik v m hematologideffectsmay bemuch more complex than was previously envisioned. It is also ckar that the examination of only one cytogenetic end point in a 11. 12. 1641. 4 13. k m e t t , D..Les, E W.,Koair. j. J. & Snyder, R - ( l m-~)j . Toxfcd. Enoiron. H d t h S,785-792. single sex may lead to a rather naive interpretationof thedata. 14. Snyder,R, Lee,E. W. & IC& J. J. (1972)Res.Cvnmun.Chum. Furthermore, the results presented here suggest that there is Pathd. P h a d . eO,191-194. no a priori reason to assume some sort of common moleculv 15. Conney. A. H..Wekh, R.,K u n h m a ~R~.., Chang, R.J- mechanism among the induction of SCE, chromosomal aber- M.. Finster, M.& Wolff. J. A (1971)Ann. N.Y. A d . sd. In, * rations, or cell cycle inhibition. The fact that male and female DBA/2 mice differed u)greatly in both the magnitude and type 155-172. 16. Lukin, S., Kuschner, M. & Drew,R. T. (1970) in In- of bone marrow response to benzene exposure or phenobarbital pretreatment (or both) suggests that thisin oitw system may be extremely valuable in elucidating which metabolic pathways are responsible for which effects Perhaps m a t important from c4rcfnogenapfp, e& Uanna, M.G.. Jr., Net* P.& G i J. R. (U.S.A.E.C. Symposium Series 18, Springfield, UBlF.- 691001, CFSTI. NBS, US. Dept.of Commerce). pp. 321- 17. T iR R, Bender,M.A. Ivett,J. L & Drew,R T. (1978)Mutot. Res. 58,293-304. the standpoint of the known leukemogenic properties of ben- 18. Tie, R., Chaillet. J. & Schneider,E. L (1975)Nature (London) zene is the observation that some of the DNA lesions induced e56,642-644. by benzene exposure may pmistthrough subsequent genera- 19. Tie, R R.,Schneider,E.L.& Rary, J. M. (1976)Exp. C d &. tions. Persistence of damage has been shown to correlate with 102,232236. carcinogenic induction in other systems (30,31).The signifi- 20. Schneider,E. L.,Sternberg, H. & Tice, R.R. (1977)Proc. Natl. cance of this finding depends on a greater understanding of A d . Sei. USA, 74,2041-2044. what types of lesions give rise to SCE and the relationship of 21. Lee, E. W., Kocsis, J. J. & Snyder, R. (19) Toricol. Appl. Phar- these lesions to carcinogenesis. m o l . 27,431-436. * 22. Kato, H. (1974)Exp. Cell Res. 85,239-247. f We thank Dr.R. Snyder for helpful discussionsand R. N.Ruffing 23. Ishii, Y. & Bender, M. A. (1978)Mutut. Res. 51,411-418. b for technical assistance. This paper was written under contract EY- 24. Kato, H. (1977)In:. Reo. Cytol. 4 9 , 5 9 7 . 76-C-02-0016 with the U S. Department ofEnergy and the Environ- 25. Shafer,D. A. (1977)Hum.Genet. 39,177-190. mental Protection Agency 26. Ikeda, M. (1964)1.Biochem. (Tokyo)53,231-243. 1. Haley, T. J. (1977)Clin. Tozicol. 11,531-548. 27. Koinuni, A., Dobaslu, Y. Tachibnna, Y.. Tsuda, K. & Kabunuma, 2. Snyder, R. & Kocsis, J. J. (1975) Crit. Reo. Toticol. 3, 265- H. (1974)I d . Health l2,23-29. I 288. 3. Vigliani, E. C. & Forni, A. (1976)Enwiron. Res. 11,122-127. '4. Dean, B. J. (1978)Mutat. Res. 47,7597. 5. Tsutsui, T., Umeda, M., Maizumi. H. & Saito, M. (1977)Cann 28. Kihlman, B. A. (1966)Action of Chemicals on D t d d h g CcUl (Prentice-Hall,Englewood Cliffs, NJ). 29. Lutz. W.K. & Schlatter. C. (1977) Ckm.-B&l. Interact. 18, 241-245. 68,609-617. 30. Goth, R. & Ra.jewsky-, M. F. (1974)Proc. Nut/. Acad. scf. USA a , 6. Davies, D. R. & Evans, H. J. (1966) in A d w w in Radiation 71, 639-643. -? Bblogy, eds AugenstemL G., Mason,R.& Zelle, M.(Academic, 31. NimU. J. W..Swann. C. F. & Peen. A. E. (1975)Nature (Londa) New York), Vol. 2, pp. 243353. 254,261-262.