Document EqY0wx81kGMKV8g9vDgEnZ3gj

EFFECTS OF BENZENE INHALATION ON MURINE PLURIPOTENT STEM CELLS E. P. Cronkite, T. Inoue, A. L. Carsten, M. E. Miller, J. E. Bullis, R. T. Drew Medical Research Center, Brookhaven National Laboratory, Upton, New York Effects of benzene inhalation on mouse pluripotent hematopoietic stem cells have been evaluated. Male mice 8-12 wk old were exposed t o 400 ppm benzene for 6 hld, 5 d l w k , for up to 9; wk. A t various time intervals exposed and control animals were killed, and cardiac blood was evaluated for changes in white blood cell (WBC) and red blood cell IRBC) content. In addition, femora and tibiae were evaluated f o r total marrow cellularity, stem cell content (as measured by the spleen colony technique), and the percent of stem cells in D N A synthesis (as determined by the tritiated thymidine cytocide technique). Exogenous spleen colonies grown from marrow of exposed animals were counted, identified, and scored by histological type. Exposure t o benzene caused significant depressions o f RBCs and WBCs throughout the exposure period, which continued for at least 14 d after exposure. Bone marrow cellularity and stem cell conrent were also depressed in exposed animals throughout the study. Tritiated thymidine cytocide o f spleen colony-forming cells was generally increased in exposed animals, perhaps indicating a compensatory response to the reduction o f circulating cells. Spleen colonies o f all types were depressed after exposure t o benzene. The significance of the reduction in cellularity, stem c e l l content, and changes in morphology o f spleen colonies is discussed i n relation t o cellular toxicity and residual injury. INTRODUCTION The biochemical and morphological changes observed in identifiable hematopoietic cells are phenotypic changes. Irrespective of the cause, they result from genotypic alterations in the pluripotent stem cell that perpetuates hematopoiesis. Although of general interest and useful for diagnosis or detection of toxicity, phenotypic changes are of l i t t l e value in understanding the mechanism o f a pollutant effect. Accordingly, in our studies of hematotoxicity we concentrate on the hematopoietic stem cell, i t s rate of proliferation and differentiation into identifiable cell lines, and The authors would like to thank L. Cook, B. Heldman, L. Honikel, N. Pappas, M. Torelli, and R. Tuthill for their excellent technical assistance. Research supported by the Environmental Protection Agency under agreement EPA-79-D-XO533 and by the US. Department of Energy under contract DE-AC02-76CH00016. Requests for reprints should be sent to E. P. Cronkite, Medical Research Center, Brookhaven National Laboratory, Upton, New York 11973. 411 Journal of Toxicology and Environmental Health, 9:411-421,1982 Copyright Q 1982 by Hemisphere Publishing Corporation 00984108/82/030411-11$2.25 412 E. P. CRONKITE ET A L the mitotic potential of the immediate descendants of the pluripotent stem cell. The pluripotent stem cell can only be assayed in the mouse by counting the number of gross spleen colonies formed in the fatally irradiated mouse following injection of a known number of bone marrow cells (Till and McCulloch, 1961). The cell forming colony is called the colony forming unit in spleen (CFU-S). The colonies are clones arising from a single cell. After several self-renewing mitoses, the colonies commence to differentiate down the red, white, megakaryocytic, or mixed cell lines. After 5-10 d, the colonies are easily seen by eye on the surface of the spleen. The hematologic phenotypic alterations induced by benzene have been reviewed (Laskin and Goldstein, 1977; Snyder and Kocsis, 1975). They indicate injury in self-renewing CFU-S that is transmitted to the identifi- able progeny. The injury may be manifested by a permanent change in genotype, in which case the phenotypic expression will be permanent, Disappearance of the phenotypic expression is indicative of either repair of the genotypic injury by processes yet to be identified or dying out of the affected clone of stem cells. Uyeki e t al. (1977) exposed mice 8 h/d to 4680 ppm benzene for 1, 3, and 3$ 8-h sessions and then measured the cellularity, CFU-S, and CFU-C content of bone marrow. Colony forming units in culture (CFU-C) are believed to be the progenitor cells of granulocytes and macrophages. They are detectable by their ability to form macroscopic colonies of granulocytes and/or macrophages in agar culture. One 8-h exposure reduced the CFU-C content of marrow to 37% of the control value the day after the exposure without any change in cellularity. The CFU-C progressively increased on d 4 and 7 but was s t i l l 23% below controls. Three and one-half 8-h exposures resulted in further decreases in CFU-C content of the marrow. Three 8-h exposures dropped the CFU-S content of marrow by nearly a factor of 3, measured 24 h after the last session. The concentration of benzene was much greater than in our studies and observations were made for only a few days, compared to our 65-d study. The studies described above involved benzene treatment schedules that bear little relation to the workplace. In this paper we present data on exposure of mice 6 h/d, 5 d/wk, at a concentration of benzene known to produce hematologic effects. The focus of our study in this initial work will be on the pluripotent stem cell, in which continuing or permanent injury to genetic control of hematopoiesis would reside. I n future studies we will address dose, time, and effect relations. METHODS AND MATERIALS Benzene Exposure Male Hale Stoner BNL mice 8-12 wk old were exposed t o either normal air or 400 ppm benzene in chambers for up to 9: wk (6 h/d, 5 EFFECTS OF BE d/wk). Expo system simila in nesting bc chamber, anc exposure per exposure ani exposure chi animals were chamber floH were generat airstream of t intervals by sampling lool tographic da vapors, using was measurec photometer. A t varioi mice were rc tized by ethe Blood counts of the expos after exposur blood cell ( Coulter elect! Zap-lsoton II cells were cc counts of ez values, the rr and WBC, 5.5 each time anc of the avcragt The hind the pooled t , 1963). Total sions. An al tritiated thyr suspension ( groups) 25 from a 250-1 0.5-mm Cu f. 60,000 nude and McCullo Their spleens colonies cou sectioned a t TE ET AL ripotent >use b y f a t a11) marrow led the arising :olonies ,tic, or on the / e been . They dentifimge in ianent. pair of of the for 1, 5, and 'FU-C) )hages. ies of ~osure le the :FU-C 1trols. :FU-C mtent ssion. s and ,tudy. i that ta on Nn to work ment udies ither 'dl 5 f I I 1 I I i 1 ! I ! 1 ! I 414 E. P. CRONKITE ET A L were identified and classified as to histological type following the criteria of Reincke e t ai. (1976). RESULTS The results reported below represent 1-47 exposures for 6 h/d, 5 d/wk, to about 400 ppm benzene. Daily averages ranged from 346 to 438 ppm, with the average concentration for any subgroup of animals ranging from 389 to 423 ppm. The cumulative average concentration for the entire series of exposures was 399 ppm. Changes in RBC counts are shown in Fig. 1. On d 11 of the study, after 9 exposures to 400 ppm benzene, there was a significant decrease in the RBC count, which remained between 54 and 80% of the control value. The oscillations may represent changes in the rate of destruction or production of the RBCs or degree of hydration of the mice. After 65 d (47 exposures) benzene exposures were terminated. Although the RBC count increased during 14 d after the termination of exposure, it was still 91% of the control value. Changes in total WBCs are shown in Fig. 2. There was a precipitous drop in the WBC count to 35% of the control value by d 4 of exposure. There were large fluctuations in the total WBC count, which are not yet understood. Following the termination of benzene exposure, the WBC count progressively increased from 38 to 80%of the control value within 14 d. 1 I I t25 --- -- BENZENE EXPOSURE SCHEDULE IO 2 0 3 0 40 5 0 60 70 TIME ( d a y s ) I 80 FIGURE 1. Serial RBC counts on mice exposed to benzene. Numbers in parentheses are numbers of mice studied. Data points are means t SE. EFFECTS OF i z 0 0 gloc fl 0 U 7: E E \ Vm 5c 3 25 FlGURE2. Tot mice studied. DE Total bc femora and since the c marrow cel. exposure da Except for of controls, values until benzene exF the control Changes shown in F pooled marr femora and there were ( and 43% up studies were Serial ch doses of (31is a close ar DNA. Thus synthesis. c ; from 26 to EFFECTS OF BENZENE O N STEM CELLS I IIII 1 0 +(L Z 0 i(4j V klO0 o\o m aIC) 75 E E \ I % 50 ? 25 1 --1 - --I BENZENE EXPOSURE SCHEDULE -y- 3 i I i i 1 i I - 41 5 i 416 E. P. CRONKITE ET A L -I gi I I I I I I I 1 81 I EFFECTS OF BE - cn I - - -W1 0 -+ -BENZENE EXPOSURE SCHEDULE z3 IO 20 30 TIM4E0 (days5)0 60 70 -- F I G U R E 3. Serial changes in total number of nucleated cells per leg (femur plus tibia) of mice exposed to benzene. Where there are t w o data points they represent separate studies. Numbers in parentheses are numbers of mice studied. Marrow is pooled from animals killed o n a given day. r7I I I I I w Y L21 I (4),BENZENE EXPOSURE SCHEDULE TIME (days) .- FIGURE 4. Serial studies of the number of CFU-S per leg (femur plus tibia) while mice are exposed t o benzene. The CFU-S assay is performed on the pooled bone marrow by injecting 6 X l o 4 bone marrow cells iv i n t o the 2 5 fatally irradiated recipients. FIGURE 5. Seri cytocide i s perfc [' HI TdR or coli to 48% by c sharp drop (66%) in D exposure, tt after the la: levels (Fig. osciIlates, b pattern wit. changes in I from benzer immature c lived rebour erythrocytic minimum o the observat Table 1 The mean maturation benzene-tre: The smaller tion, the srr cells to a m The eff I I nice rs in I I I I 1 1 I L ! ! I ! i ! sed Ime f i 418 E. P. CRONKJTE ET A L zr E! VI W Id6f J l 125;) I I I1 UNDIFFERENTIATED AND IMMATURE ERYTHRO AND GRANULOCYTIC COLONIES I 1 iI I 1=!E &- +!-BEN-ZEN-E E-XP,O-SURE SC-HEDUL-E t -;To -&O' I O 20 30 60 TIME (doys) F I G U R E 6. Serial studies on histological types of coionies present in s p l c ~ nsections ( H & E stain). Data are expressed as percent of control value at each point. Ten spleens from controls and benzene-xposed mice were studied for each data point. and control mice with cytocidal concentrations of [ H ] TdR are shown in Table 2. Incubation of control bone marrow with [ 3 H ] T d R resulted in a reduction of the number of spleen colonies by 28%. When marrow from benzene-treated mice was incubated in the same manner, a 49% reduction in spleen colony number was observed. Thus the fraction of CFU-S killed by [ 3 H ] T d R was 28% in controls and 49% in benzene-treated mice. The TABLE 1. Projected Area o f Splenic Colonies' Produced by Bone Marrow Cells from Benzene-treated and Control Mice Group Number of spleens UNE Size of colony in each typeb!' (mm') ERU ER ERM Control 6 1.71 5 0.42 (n = 6) 2.32 f 0.35 (fl = 39) 0.75 f. 0.13 (n = 5 ) Benzene-exposed 6 1.28 (n = 1) 0.90 f. 0.13 (n= 7) 1.31 +_ 0.20 (n = 16) 0.42 (n = 2) 'Histological colonies from middle saggital sections were traced and size was computed by the two diameters measured. bUNE, undifferentiated with trace of erythropoiesis; ERU, erythropoietic immature; ER, erythropoietic a l l stages of maturation; ERM, erythropoietic mainly nondividing [classification from Reincke e t al. ( 1 9 7 6 ) j . 'Mean f SE. EFFECT TABLE '2 Is Derive( Grc Control Benzene- 'The si small nun ~ E Re,r C~ nondividi ~ CFU-S have n from k of all compar DI! Gil pluripc hid, 5 50,000 in bon CFU-S decrea: strikini d of e> al. rep decreas we ob5 transfu to ben. with c progres exposu Alt examin Their L TABLE 2 Number of Different Types of Spleen Colonies Seen in Spleen Sections When Bone Marrow Is Derived from Benzene-treated and Control Mice, Each Subjected to [' H I T d R Cytocide Group Total N u m b e f of colonies +Undifferentiated ER GR' ERM CGRM' ( H & E stain). ;ontrols and shown in ilted in a 'ow from -eduction I-S killed 7ice. T h e Is from - M 0.1 3 1 -i I by the ire; ER, issifica- 420 E. P. CRONKITE ET AL tetraploid cells and a reduction in the specific activity of [3H]TdR in DNA, indicating an aberration in continuing cell proliferation and the cell cycle. These changes would lead to a reduction in the number of cells produced by the bone marrow. Their studies also indicated that benzene affects the amplification of the stem cell progeny during the presence of benzene and i t s metabolites. Since there are populations of cells with short transit times in the marrow, these effects do not explain sustained hematopoietic effects after removal of benzene and i t s metabolites. The cause of long-term effects after benzene exposure must be sought by study of stem cells. The substantial decrease in absolute marrow cellularity in our studies can only be explained by a large reduction in the amplifying populations of identifiable erythrocytic and granulocytic precursors, since other cell lines account for but a small fraction o f the bone marrow cells. The size of these transit populations can be reduced by diminished input from unidentifiable precursor cells (CFU-S and intermediate early progenitors) or by diminished amplification. The elimination of a terminal mitosis would decrease the population of nucleated cells by a factor of 2. A reduction in input from the stem cells by a factor of 2 with constant amplification would also reduce the marrow population within one transit time through the marrow by a factor of 2. The reduction in absolute number of CFU-S in the marrow was greater than a factor of 2. Production of CFU-S was increased, as evidenced by a larger fraction being in DNA synthesis. The number of stem cells produced is directly proportional to the number in DNA synthesis. On d 11, for example, control bone marrow contained 58 X l o 6 cells per leg and marrow from benzene-exposed mice had 20 X lo6 cells per leg, a reduction by a factor of 3. The CFU-S content is 17.2 X l o 3 per leg in controls and 3.3 X l o 3 in benzene-exposed mice. The number of CFU-S in S phase for controls was 3.61 X l o 3 (17.2 X l o 3 X 0.21) and for benzene-exposed mice was 1.55 X l o 3 (3.3 X l o 3 X 0.47). If the DNA synthesis time is the same in both groups, the production rate of CFU-S in benzene-exposed mice is benzene-exposed mice contained 53 X l o 6 cells, compared to 52 X lo6 cells in controls. The respective CFU-S were 14.1 X l o 3 in controls and 5.8 X l o 3 in benzene-exposed mice with 16 and 47% in DNA synthesis, respectively. Hence, there are 0.16 (14.1 X l o 3 ) or 2.26 X lo3 in S phase in controls and 0.47 (5.8 X l o 3 ) or 2.73 X lo3 in benzene-treated mice. I n order to reconstitute the marrow cellularity by d 50, there must have been a greater production of CFU-S at earlier intervals and/or an increase in the amplification of the transit populations. The problem is of more than trivial interest since others have described hypercellularity during benzene exposure and/or a failure to see a diminution in the marrow celIuI arity. EF it ce re: rh cc ce Be Gi Irc La La Re Sn St TI Ut I TAL. R in cell cells zene e of hort ined The .udy ;dies ions cell size rom ors) osis I. A tant nsit lute . 2. 2ing ctly Pk .om :tor 103 rols was : in 3 is -om 106 and Isis, lase ice. ave ase ore ing ow EFFECTS OF BENZENE ON STEM. CELLS 421 In this and other toxicological studies on stem cells in this laboratory, it is evident that the morphology of splenic colonies produced by stem cells from radiation or chemically treated hosts i s a useful guide to residual injury. Disintegration, dispersion of cells, pyknosis, and karyorrhexis are seen to a slight degree in colonies produced by stem cells from control mice. The incidence of these is greatly increased when the stem cells are from irradiated and/or chemically treated mice.