Document 37JzgZkJ6mQek8gZZwRMQeva3

Exd. Hernatol. I 7:300-393 f 1989) I989 International Societv for Expenmcntal Hematology T h e Hematopoietic Stem Cell Compartments in Mice during and after Long-term Inhalation of Three Doses of Benzene H. J. Seidel. Edda Bashel. and D. Zinser Institute for Occupational and Sucial 1Mediclne. Ilniversity of Ulm. FRG (Received 2 I J u n e 1988: in wvrsed.form 23 September 1988: accepted I 4 October 1988) Abstract. Female BDF, mice were exposed for 16 weeks to airborne concentrations of 100. 300, and 900 ppm of benzene, 6 h per day, 5 days per week. Bone marrow hemopoietic stem cell companments and peripheral blood cell counts were studied using clonal assays and standard methods. Dosedependent depressive effects were observed on all stem cell compartments. Only the erythroid colony-forming units (CFU-E) compartment was depressed during exposures to 100 ppm; CFU-E were more sensitive than the erythroid burst-forming units (BFU-E), spleen CFU (CFU-S), or G-M CFU (CFU-c) during exposure to 300 ppm or 900 ppm. Lymphocytopenia was observed in the peripheral blood. After benzene-free intervals, a regeneration of lymphocyte numbers a n d slow normalization of stem cell numbers was seen. Complete recovery from the 16 weeks exposure to 300 ppm was seen between 73 and 185 days. - -Key words: Hemopoietic stem cells Mice Benzene inhalation Based on epidemiological data benzene is considered a human carcinogen [l-31. Until recently there have been uncertainties whether the carcinogenicity could be demonstrated in experimental animals [4]. Inhalation studies with mice have shown development of tumors in a high percentage of exposed animals [ 5 ] . The toxicity of chronic benzene exposure to hemopoietic stem cells and the effects on some functions of the immune system have also been described [6-121. The doses used in the previous laboratory studies varied between 10 and 4000 ppm. Our research program with benzene focused on the different hemopoietic stem cell types and addressed questions related to long-term exposure to concentrations of 100, 300, and 900 ppm. The objectives were to identify the most sensitive parameter and to determine if a dose-response effect could be demonstrated. Materials and methods .4nima/.v. Female mice (21-25 g) were used (C57B1/6x DBA/Z F, hybrids). These were obtained from the Zentralinstitut fur Versuchstierzucht. Hannover. FRG. They were fed commercial pellets and water ad libitum. During the benzene exposure period of 6 h, no food was made available. The mice were kept under controlled climatic conditions in the Central Animal Facility of the University of Ulm. 4ddrPss rrpnnr rrquests fo:Dr. H.J.Seidd, Institute for Occupational md Social Medicine. University of Ulm. Oberer Eselsberg. D 7900 Ulm/Donau. FRG. Inhalciion e.uposure. Benzene vapor was generated by forcine 5::- air through a box with a constant influx of benzene. The air <;.a3 ':*.containing benzene was mixed with a secondary Row ofair to q n w thedesired mixtures. The inhalationchambersprovided 29 ::- per Xour. BenLene concentrations in the chamber werc twice daily by gas chromatography as described by Zinsc :o.m.. .'.? the variance was <5Oh deviation from the desired conL.-:.::& The mice were exposed to the benzene mixtures for 6 h per a! f days per week (Monday through Friday). Control mice were h o w in the same chambers without admixture of benzene. Hematological techniques. Blood samples were obtained from &* axillary plexus after ether anesthesia. Total numbers of white b l m cells were determined using an electroniccell counter (CoulterC o m e ZF). Differential counts of blood smears and the reticuloqTe couno were obtained using standard methods. Organ weights of the s p i m and the thymus were also determined. Bone marrow cells from the femur were flushed out ints a itnova volume of medium, suspended in a single-cell suspension. .--::tcQ and subsequently used for the stem cell assays. Spleen co! - h g units (CFU-S) were determined using 800-rad imdir;: .:as ~ ients (x-rays, 280 kV, 12 mA with 1.5 mm Cu and 1.0 mc: 31 a filter, focal distance ?O cm, 30 radmin). Macroscopic spleen colnnio were counted at day 9 without further characterization. Erythroid burst-forming unit (BFU-E),erythroid colony-icrmlq unit (CFU-E),and G-M CFU (CRJ-C) cultures were establishcC :x methylcellulose using standard protocols as described in pre\.iou communications from this laboratory (see Seidel and Opitz [la]JT.J Seidel and Kreja [ 151. Cultures were established using feu1 calf fir horse serum. Mixed-lymphocyte-culture supernatant was used JS 3 source of burst-promoting activity for the BFU-E cultures. Trsrendotoxin serum from mice was used as a source for colony-stin- ulating activity in the CFU-C cultures. Sheep erythropoietin VC? i i i was used for BFU-E and CFU-E cultures. All colony cou- : -.:re convened into colony numbers per femur. Each day contrci. . -:a were set UP using the pooled femoral m a m w of three conti':; mmals. At all experimental points four benzene-exposed micc \\ere tested individually and their data were compared with those of he controls. Unless indicated otherwise, the assays were done on S h day mornings, Le.. about 66 h after the end of the exposure pefid of the preceding week. For the data summarized in the tables and hgures. the colony numbers of the controls were set as 100%. The absolute numbers per femur were as follows: CFU-S -5000, C K - C -28,000. B R I - E -8000. and CRI-E -30,000. Results Stein cells during rhe e-vposlrreperiod The rclative numbers of BFU-E, CFU-E, and CFI femur in mice exposed to 100 and 300 ppm for 16 - . A S and 10 900 ppm for 8 weeks arc shown in Figure I.Th'rC was considcrable variation. Table I summarizes the data. 4 dosc-rcsponse rclationship can be scen. especially afrcr 5 wecks ofcxposurc for the BFU-E and CW-E. Also. the CFVE compartment sccms to be more sensitive than the B n 1 - E and the CFU-C compartments. This is especially the case '/ FEYUR FEMUR -$:& 1. BFU-E, CN-E,and C N - C numbers per femur (as percent ~ d c o n u o l si)n individual mice exposd to 100 pprn (A), 300 ppm -.%.--,...... and*90)0 p.pm (0)of benzene. ,$??: ..--& Za: h- rthe mice exposed to 100 ppm, for which no effect was '.-.2.-_. hwith the BFU-E at least up to 10 weeks. The CFU-E '..ti.-,.. below 60% from I week onwards. The CFU-C com. -9ulment does not seem to be affectedas much as the CFLJ- .c-...E..The same holds true for CFU-S (Table 1). For the group :?;.*a 300-ppm exposure. after 13 weeks the CFU-E num- -:7-;b.Aa'swereabove the control level for three out of four animals. *.3 $ a m cells after the exposure period 5;- ::After an exposure period of 8 weeks animals were evaluated .;-be*nzene-free intervals. Figure 2 presents the BFU-E and W - E determinations. Ten and 17 days after 100-ppm ex- I: I *I...UIII.~II..~..I~~~ days Fig. 2. BFU-E (closed symbols) and CFU-E (open symbds) n m - ben per femur (as percent of controls) in individual mice after previous exposure over a penod of S weeks to 100 pprn, 300 pprn. and 900 ppm, respectively, of benzene. Lines combine mean values. I ! I I I posure, the B N - E were above control values and the CRI- E below control values, although normal levels were seen at day 3 (66 h after the end of the exposure). After an exposure to 300 and to 900 ppm, a slow regeneration of the stem cell numbers was seen. The B N - E data at day 24 after a 300pprn exposure, and at day 19 after a 900-ppm exposure, seem to indicate that a steady state was not reached after this time period. The stem cell recovery after 300 ppm of benzene exposure over a period of 16 weeks is shown in Figure 3. There was a wave of regeneration with an overshoot of CFU-E during the first 3 weeks in a normal atmosphere. Most BFU-E and C N - E numbers remained below controls at least for 73 days: a complete normalization was achieved by day 185. Table 1. z - Stem cell numbers in femurs of benzene-exposed micc as percent of controls (mean f SD) BFU-E 4 weeks CFU-E BRI-E 100 ppm 300 ppm 900 ppm 88% t 13% 88% f 14% 66% ? 25% 48% r 8% 35% f 18% 35% f 10% *96% t 17% 55% 22% 32% t 8% CFU-S CFU-c CFU-S 100 ppm 300 ppm 900 ppm 92% f IIYO 70% f 19% 82% f 17W 9496 t 8% 59% t 17% 6396 +. 24% ND 65% -c 15% ND 8 weeks CFU-E 99% t 28% 73% f 30% 35% r 2% CFU-C 103% 2 15% 47% _c I 3% 5 I % 2 17% 302 %3 BFU- E 'FZYLIR 0 C.W-E '.'IpYLIR 3 Fig. 3. BRJ-E and CFU-E (upper part) and CFU-C (lower part) numbers per femur (as percent of controls) in individual mice after previous exposure over a period of 16 weeks to 300 ppm of benzene. Lines combine mean values. Peripheral blood A slight anemia developed in mice exposed to 300 and to 900 ppm of benzene. After 4 weeks the erythrocyte count was 7.4 x 106/mm3and 7.6 x 106/mm3as compared with 9.5 x 106/mm3in controls. The anemia did not develop further during the next weeks. With 300-ppm exposure a slight recovery, even during the benzene exposure period, was seen to reach 8.6 x lO6/mm3at 13 weeks and also at 16 weeks. The reticulocytes showed considerable variation. Three out of four animals showed a reticulocytosis at 13 weeks. The same animals had elevated numbers of C N - E in the marrow (see Fig. 2). Also, three out of four animals had elevated reticulocyte numbers 2 weeks after the end of the 16-week exposure period. The number of granulocytes was not affected by the benzene exposure. The number of lymphocytes showed a dose-dependent reduction. As shown in Table 2, there were high lymphocyte numbers in the control group of the 300-ppm experiment, which had been performed 1 year earlier. There is no explanation for this difference. With a 100-ppm exposure, the number of lymphocytes is normal immediately after the end of the exposure (66 h; see above), but there is a decline after 1 or 2 weeks in a normal atmosphere. The effect on the lymphocytes in the 300- and 900-ppm groups was reversible; in most instances a benzene-free interval of 1 or 2 weeks was enough for complete regeneration or even an overshoot. Discussion There are several reports of studies on hemopoietic stem cells and peripheral blood in benzene-exposed mice. These studies showed substantial effects. A dose-dependent CFU-S reduction after 10 exposure days (6 h per day) was reported by Cronkite et al. [9]with doses up to 400 ppm that were inversely related to the increase of CFU-S in DNA synthesis. Gill et al. [7]reported a depression to about 20% after 4000 ppm over a period of 6 weeks. Depressed CFU- -Expenmental Hematology \'Ql, , . .> Exposure penod . - _ _Time in norma1--\ Control 100 ppm Control 3CO ppiri Control 900 pprn 4 wccks 2990 2 1 IjP 2690 -ir I580 4800 2 2000' 2230 3 380 2570 f 750' 1270 f 590 - -1869 z 3-0 3130 = 900 7390 1. 1070 -- 1: - \: 3 - , , : ,yn - 4010 ::'.*; Control 8 weeks 3170 f 1700' 1 week - - -2 ~ C c U 100 ppm 3510 2 1170 1780 C 240 2080 = ?.!: Control 6500 2 1000' - 300 pprn Coptrol 906 ppm 2170 c 270 3070 2 820' 1250 -+ 4QO 3370 z 500 3660 3 1670 3699 z $891 - -1 . .::3 Peripheral blood lymphocyte numbersin mice exposed ;L . . a s centrations of benzene, studied at the end of the exposux ; c c r ~ and after I and 2 weeks in normal atmosphere. Numbers obtained from three groups ofcontrol micc immeaiawiv after the exposure period (4 and 8 weeks. respectively) and I a~ 2 weeks la!er. Their numbers were matched (n = 9). Mean v a l w & SD. C numbers were reported by other researchers [6. 7. IO]. Hilderbrand and Murphy [ 101 presented BFU-E and C K E data for an exposure to 4062 ppm over 14 days. ;- :?ar study an initial depression of CFU-E (deeper tha;: . ' .-E levels) was observed however, during the benzene ~X;JWC the C N - E rebounded to slightly higher levels than thc con- trol values. This might, in principle, be the same phencmenon as seen in our study for a benzene exposure of 300 p p n at 13 weeks. The lowest concentration of benzene that was studied was 10 ppm. This was for an exposure period of 179 days conducted by Baarson et al. [ l 11. They reported 3 depression of CFU-E below 5% of controls but not of B K E. It must be noted that these studies were different with respect to the strain of mice used. in vitro techniques uscd. and also doses and time intervals studied. Our preser:: >:A!. will allow a better evaluation and comparison of the a. - 2s effects,because three different assay systems (four when i F C S were induded) were used simultaneously. Identical prctocols in the three experimental runs were used. We concluded that the erythroid system, as represented by the BFU-E and the CFU-E in the marrow and the erythrocytes in the peripheral blood, is more sensitive than the m?- eloid cell system. This holds true when evaluations are made using CFU-C numbers and peripheral blood neutrophils. Within the erythroid system the CFU-E are the most sensitive-in agreement with previous reports cited above. There are. however. important observations that warrant P. '. .'r investigation. The high CFU-E number observed late c '1 the exposure period, accompanied by a reticl;locytosis. 2 .!I be explained. Also. our observation that the anemia did nor worsen during the exposure periods may indicate that the erythroid cell system can adapt to the benzene; this should be evaluated. This aspect will be described in future communica tions. ?T-- FZ- F -:-=x-_-r. . ,-e* J d-t- *del e[ al.: Hemopoietic Stem Cells after knzene Exposure ..bfie experiment with 100 pprn, a depression of CFU-E was seen as the principal effect of benzene exposure. s ! w e k s of benzene exposure with a subsequent short f@lm to periods of normal air. an overshoot of BFLJ-E and a lymphocytopenia were observed. This may &le the presence of regulatory processes that shouid be #I&. The other effects observed on lymphocyte n u m - ;n agreement with the data of Cronkite et al. [SI. .- doses used in our studies are high when compared m a n exposure data and threshold values suggested I(ijmous organizations (see [ 161). Therefore, concentrations &la, ppm should be studied. The C N - E data of Baanon :.+%,~& [I I] for IO-ppm exposures and other experiments with I a d rats utilizing cytogenetic methods indicate that -+.-can b e expected [ 171.It should be possible to c o m p a r e .a-%Jtnsitivity of the various cell compartments a n d evaluate .e.. ychniques for their appropriateness in establishing a "no ;anlevel for benzene exposure. =&soy M. Erdem S, Dincol G (1974) Leukemia in shoe workers '..aposcdchronically to benzene. Blood 44337 S I n f i n t e PF. Rinsky RA,Wagoner JK, Young RJ (1977) Leu-> kemia in benzene workers. Cancer 276 Rinsky RA.Young RJ. Smith AB (1981) Leukemia inbenzene iortrm. Am J Ind Med 2:217 Henschler D (1987) Risk assessment and evaluation ofchemialcarcinogens: present and future strategies. Cancer Res Clin Onml 113:l a n k i t e EP, Drew RT, Inoue T, Bullis JE (1985) Benzece bsmatoroxicity and leukemogenesis. Am J Ind Med 7:447 Uyeki EM, Ashkar AE, Shoeman DW, Bise1TU (1977) Acute 303 toxicitv of benzene inhalation in hemopoietic precursor cells. Toxicol Appl Pharmacol 40:49 7. Gill DD. Jenkins VJ. Kempen RE. Ellis S f 1980) The importance ofplunpotcntial stem cells in benzene toxicity. Toxtcoiog: 16.163 8. Green JD. Snyder CA. Lobue J. Goldstein BD. Albert R E ( 1 98I) Acute and chronic dose-response erfects niinhalcd benzene on multipotential hematopoietic stem (CFrJ-S)and granulocytc-maaophage progenitor (GM-CFU-C)cel!s in CD- I mice. Toxicol Appl Pharmacol 58:492 9. Cronkite EP. Inoue T. Carsten AL, Miller ME. Bullis JE. Drew RT ( 1 982) Erects of benzene inhalation on murine pluripotent stem cells. J Toxicol Environ Health 9:411 0. Hilderbrand RL, Murphy MJ Jr (1983) The effects of benzene inhalation on murine hematopoietic precursor cells (CFU-e. CFU-gm). Int J Cell Cloning 1:240 I. Baarson KA. Snyder CA, Albert RE (1984) Repeated exposure of C57 BL mice to inhaled benzene at 10 ppm marked by depressed erythropoietic colony formation. Toxicol Lett 200:397 12. Xozcn MG, Snyder CA. Albert RE (1984) Depressions in Band T-lymphocyte mitogen-induced blastogenesis in mice exposed to low concentrations of benzene. Toxicol Lett 20343 13. Zinser D. Pforr M, Seidel HJ. Riedner TM (1987) Chronische Benzolexposition in einer Inhalationskammer: Technische und methodische Vorausscuungen. Verh Dtsch Ges Arbciumed. Stuttgart Gentner Verlag. p 653 14. Seidel HJ,OpiU U (1979) Erythroid stem cell regeneration in normal and plethoric mice treated by hydroxyurea. Exp Hema- to1 7:500 IS. Seidel HJ,Krcja L (1981) Role ofBPA and erythropoietin for BFU-E growth from Friend virus infected mice. Stem Cells I: 367 16. Deutsche Fonchungsgemeinschaft, Ed (1987) Maximale Ar- beitsplatrkonzen trationen und biologischeArbeitsstofftoleranzwerte. Weinheim: Verlag Chemic 17. Erexson GL. Vilmer JL, Steinhagen WH,Kligennan AD ( I 986) Induction of cytogenetic damage in rodents after short-term inhalation of benzene. Environ Mutag 8:29 I I i ! I