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The detection of in vivo hematotoxicity of benzene by in vitro liquid bone marrow cultures
TOXlCOLOGYANDAPPLIEDPHARMACOLOCY60,346-353
(1981)
The Detection of in Wvo Hematotoxicity of Benzene by in vitro Liquid Bone Marrow Cultures’ KENICHI
HARIGAYA,*
MARILYN, E. MILLER, EUGENE AND ROBERT T. DREW
Medical Research Center, Brookhaven National
Laboratory,
P. CRONKITE,’
Upton, New York 11973
Received January 27, 1981: accepted April 24, 1981 The Detection of in Vivo Hematotoxicity of Benzene by in Vitro Liquid Bone Marrow Culture. HARIGAYA,K.,MILLER, M.E., CRONKITE,E. P., ANDDREW,R.T.(I~~I). Toxicol. Appl. Pharmacol. 60, 346-353. Bone marrow toxicity induced by benzene inhalation was investigated in mice using an in vitro bone marrow culture system and a bioassay for ‘leukemia. Donor animals (6-week-old female C57Bl/6J) were exposed to 400 ppm benzene 6 hr/day for either 9 days (5 days/week) or for I I consecutive days. Cell suspensions from long-term bone marrow cultures established from benzene-exposed mice showed a progressive reduction in the number of spleen colony-forming cells (the hematopoietic stem cell) compared to control. Different combinations of adherent cell layers and reinoculated cells showed that cultures containing bone marrow cells from benzene-exposed mice had a lower capacity to maintain stem cell proliferation than normal combinations irrespective of whether benzene-exposed marrow was in the adherent cell layers or reinoculated cells. These results indicate that benzene inhalation produces stem cell injury leading to diminished self-replication and derangement of the adherent marrow population. Bone marrow cultures from benzene-treated mice did not develop detectable transformation in vitro throughout the culture period. Neonatal mice inoculated with cultured cells from benzene-exposed mice have not developed leukemia during an S-month period after inoculation.
A continuous liquid culture system of bone marrow has facilitated the study of the proliferation and differentiation of hematopoietic stem cells and their cellular environment (Dexter et al., 1977b). In this mouse in vitro system, hematopoietic cell lineages consisting of granulocytes, monocytes, macrophages, megakaryocytes and erythrocytic and lymphocytic precursors are generated (Dexter et al., 1977b; Dexter, 1978; Testa and Dexter, 1978; Williams et al., 1978b; ’ Research supported by the Environmental Protection Agency under Agreement EPA-79-D-X0533, and the U.S. Department of Energy under Contract DEAC02-76CHOOO 16. 2 Present address: Department of Pathology, Keio University School of Medicine, Tokyo, Japan. 3 To whom reprint requests should be addressed 0041-008X/81/1
10346-08%02.00/O
Copyright 0 1981 by Academic Press. Inc. All rights of rcproduclion in any form reserved.
Johes-Villeneuve et al., 1980). There are two basic components in this culture system. The first is a cell population which adheres to the culture flask and is composed of phagocytic mononuclear cells, fat cells, endothelial cells, and epithelial cells. The second is a cell pop ulation suspended in the culture medium which when examined by light microscopy is found to contain mainly granulocytes and macrophages. The latter cell population is conveniently referred to as the nonadherent cell population. Spleen colony-forming cells (CFU-s, hematopoietic stem cells), are routinely maintained for at least 12 weeks using mouse bone marrow in this culture system (Dexter et al., 1977b). Some CFU-s in the nonadherent cell population arise from the adherent cell layers during the culture period 346
HEMATOTOXICITY TABLE MEASURED
CONCENTRATION EXP~WRE
1 OF BENZENE
IN THE
CHAMBERS
Expt groups”
Days of exposure
Benzene average concentration (mm * SW
Range of daily averages (ppm)
IP
9
411 f 20
392-446
IR
9
403 * 25
384-461
HP
11 9
376 -+ 21 392 + 13
348-408 374-408
IIR
11 9
410 +- 19 396 2 I
382-443 387-410
a I, Experiment I; II, Experiment II; P, benzene exposure of donor animals for the establishment of adherent cell layers; R, benzene exposure of donor animals for the reinoculated cells.
(Mauch et al., 1980). The spontaneous decline in stem cell replication appears to be attributed to the environment of the adherent portion of the marrow culture rather than to exhaustion of self-renewal potential in this bone marrow culture system (Dexter et al., 1977b; Williams et al., 1978a). The close relationship between chronic high-level benzene exposure and the occurrence of leukemia in humans has long been recognized (Brief et al., 1980, Snyder et al., 1977). Cronkite ( I96 1) reviewed benzene toxicity of man and concluded that any agent which produces marrow aplasia is a putative leukemogen. There have been reports of benzene-induced leukemia in mice (Lignac, 1932; Maltoni and Scarnato, 1979; Snyder et al., 1980). However, other investigators failed to demonstrate a relationship between exposure to benzene and the subsequent development of leukemia in mice (Thorpe, 1974; Ward et al., 1975). At the present time benzene exposure of man is associated with the development of leukemia but whether benzene is an initiator or promotor is not known. Benzene is unquestionably a myelotoxic substance which affects undifferentiated and more.differentiated-hematopoietic cells in mice (Lee et al., 1974;
347
OF BENZENE
Snyder et al., 1977; Uyeki et al., 1977; Gill et al., 1980). In this study, the mechanisms of myelotoxicity and leukemogenicity were investigated in mice after acute benzene inhalation by using a liquid culture system of bone marrow and a bioassay for leukemia (Gross, 1970). Our studies indicate that both the injury of hematopoietic stem cells and the derangement of their cellular environment are important in the myelotoxicity of benzene. Leukemogenicity has not been demonstrated 8 months after the inoculation of neonatal mice with cultured cells from benzene-exposed mice. METHODS Benzene exposure. Exposures to benzene were carried out in an isolation chamber system similar to that described by Laskin et al. (1970). Animals were housed in nesting boxes in the living quarters of the chamber and were allowed food and water ab libirum, except during the actual exposure periods. The room was maintained on a 12-hr light cycle. During exposure animals were placed in wire cages and then into a 128-liter exposure chamber mounted inside the isolation system. Control animals were housed in similar cages for exposure to air only. The chamber was operated at approximately 15 air changes/hr. Benzene vapors were generated by metering air through a bubbler and then into the airstream of the inhalation chamber. Concentrations were monitored at halfhour intervals using an automatic gas sampling coupled to a gas sampling loop in a Packard Model 417 gas chromatograph. The chroTABLE MEAN
Weeks after reinoculation 0 1 3 5 8 10 13
No.
2A
OF SPLEEN
COLONIES
IN RECIPIENT
MICE
Control 18.3 f 2.2 4.0 + 0.8 4.3 _+ 0.6 16.6 I! 2.1 Confluent’ 8.1 f 1.0 12.9 -+ 1.8
(CFU-s)
Benzene 7.6 2.9 3.3 1.6 1.3 0.4 0.2
f + 2 * + + f
1.2 0.5 0.6 0.3 0.4 0.3 0.2
Note. 10’ nucleated cells inoculated. Mean + SEM, n= IS. ’ Colonies too numerous to count (>30).
HARIGAYA
348 TABLE
and the contents of one femur and tibia were each flushed into a screw-capped. 25-cm* plastic culture flask (Corning Co., Corning, N.Y.) (one mouse yielding material for two flasks) containing Fischer’s culture medium (Grand Island Biological Co., Grand Island, N.Y.), supplemented with 25% horse serum (Flow Lab. Inc., McLean, Va.), antibiotics (100 U/ml penicillin and 100 pg/ml streptomycin), and IO-’ M hydrocortisone sodium succinate (Upjohn Co., Kalamazoo, Mich.). No attempt was made to obtain a single-cell suspension. The cultures were gassed with a mixture of 5% CO* in air, incubated at 33’C, and were fed once or twice a week by removal of half the growth medium and addition of an equal volume of fresh growth medium. After 3 weeks, all nonadherent cells were removed by decanting and the adherent cell layers were washed twice with Fischer’s medium. At this time a second group of 24 benzene-exposed mice was killed, the femurs and tibias were removed, pooled, and ground with a mortar and pestle. Single-cell suspensions were prepared by passing the suspension through successively finer gauge needles (18-27). and the cell concentration was determined by a Coulter electronic particle counter. Ten million freshly isolated cells were added in fresh medium to the adherent cell layer. This method of cell preparation was used for reinoculation in order to increase the cell yield, as well as to shorten the preparation time. Feeding of the cultures continued once or twice a week by removing half the growth medium and adding an equal volume of fresh growth medium. The suspensions removed from each flask were pooled, centrifuged at 8OOg for IO min. and then washed twice in Hepes-buffered minimal essential medium (MEM) (GIBCO). At appropriate time intervals, CFU-s and leukemogenicity in neonatal mice were determined using the pooled cell suspensions which were kept on ice during processing. Final cell suspensions were adjusted to appropriate cell numbers for CFU-s assays (I X 106/ml) and neonatal injection (2 X lO’/ml).
2B
TOTAL NUMBER OF CFU-S AND NONADHERENT CELLS IN SUSPENSION PER FLASK IN THE BONE MARROW CULTURES No. of nonadherent cells/flask (X IO’)
No. of CFU-s/f?ask Weeks after reinoculalion 0 I 3 5 a IO I3
COllld 1833 105 194.9 199.2 >480 48.9 38.7
BenZCne
(5.5) (10.6) (10.9) (226.2) (2.7) (2.1)
760 70.3 149.9 25.6 6.6 I .4 Cl
(9.9) (19.7) (3.4) (0.9) (0.2)
CO~l~Ol
Benzene
IW 2s 45 12 I6 6 3
100 24 45 I6 5 3 3
ET AL.
’ Percentage of initial inoculum of CFU-s. 34 flasks in each point.
matographic data were confirmed by wet chemical analysis for benzene vapors in the chamber using a bubbler with ethanol as the absorbing solution. The absorbance was measured at a wavelength of 254.6 nm on a uv spectrophotometer. Groups of 25 C57Bl/6J female mice (BNL) were exposed to either normal air or 400 ppm of benzene in chambers for either 9 days (6 hr/ day, 5 days per week) or 11 consecutive days (6 hr/day). The nominal concentration of benzene throughout these experiments was 400 ppm. The average measured concentration for any one experiment did not vary from that by more than 6%. The highest and lowest measured daily averages were 461 and 346 ppm, respectively (Table 1). Bone marrow cultures. On the day following the last exposure, mice were killed by cervical dislocation and dipped in 70% alcohol. Whole legs were removed, freed from muscle and kept in sterile plastic plates on ice until processed. One end of the bone was cut with scissors, TABLE
3
DIFFERENTIALS (%) OF CULTURED CELLS IN SUSPENSIONS Monocytes and macrophages
Granulocytes
Others”
Immature cells
Weeks after reinoculation
Control
Benzene
Control
Benzene
Control
Benzene
Control
Benzene
I 3 5 8 IO 16
66.5 64.0 26.0 1.0 39.0 75.0
73.0 58.5 29.0 3.5 3.5 0
17.5 33.5 72.0 98.5 44.5 18.0
13.5 39.0 69.5 96.5 96.5 100.0
14.0 2.5 1.5 0.5 13.5 7.0
13.5 2.0 I.5 0 0 0
0 0 0 0 3 0
0 0.5 0.5 0 0 0
’ Megakaryocytes or undetermined cells.
HEMATOTOXICITY TABLE FIVE DIFFERENT CELL LAYERS AND
4
COMBINATIONS REINOCULATED
OF ADHERENT BONE MARROW
CELLS
1. II. III. IV. V.
No. of flasks
Adherent cell layers
8 8 8 8 8
N N B B 8’
Reinoculation” (No. of cells to reinoculate) N (0.45 B (1 x N (1 X B (1 x B’ (1 x
x 10’) 10’) 10’) 10’) 107)
a N, Normal control bone marrow cells; B, bone marrow cells from mice exposed to benzene for 11 consecutive days (6 hr/day); B’, bone marrow cells from mice exposed to benzene for 9 days (5 days/week, 6 hr/day). Donor animals were maintained in room air for 5 days after the cessation of benzene exposure. Spleen colony assay. The spleen colony assay (Till and McCulloch, 1961) was performed in female C57BL/ 65 mice 8 to 12 weeks old. Groups of 12 to 15 mice were injected iv with 1 X IO5 cells 3 hr after 730 rad of whole body irradiation administered by a 250 kVp General Electric Maxitron X-ray machine operating at 250 kVp and 30 mA (target distance, 60 cm; 0.5 mm copper plus 1.0 mm aluminum filter; dose rate, 103-108 rad/min). The endogenous colonies were determined in animals receiving only Hepes-buffered MEM. Spleens were harvested 7 days later, and colonies were counted at 3X magnification after at least 24 hr fixation in Bouin’s solution. The endogenous spleen colony count was subtracted from the spleen colony counts obtained from mice injected with bone marrow cells. Leukemogenecity in neonotal mice. Newborn (less than 48 hr old) C57B1/6J mice, inoculated ip with 5 X IO5 cultured cells from benzene-exposed or control mice, were weighed and evaluated for splenomegaly and death weekly after weaning. Microhematocrits and morphological examination of peripheral blood were performed monthly.
RESULTS On the day following the last exposure, the femur and tibia from a single mouse were flushed into an individual culture flask to establish the adherent cell layer (40 flasks). Single cell suspensions were prepared by flushing femurs and tibias from four sepa-
OF BENZENE
349
rate mice and immediately testing for CFUs content and leukemogenic potential in neonatal mice. After 3 weeks, a second cohort of mice treated identically were killed and single-cell suspensions of bone marrow were prepared by grinding marrow with a mortar and pestle. The single-cell suspensions were assayed for CFU-s content and leukemogenicity. An aliquot of the pooled, single-cell suspension was added to each of the established bone marrow monolayer cultures (1 X lo7 nucleated cells/flask). There were no morphologic differences between the adherent cell layers formed from benzene-treated and normal mice when examined with the inverted microscope. The culture suspensions were examined for CFUs and leukemogenicity at appropriate times during the culture period. There was a 30% reduction in the cell counts of bone marrow cell suspensions from benzene-treated animals. There were marked differences in the CFU-s frequencies between bone marrow cells from benzene-exposed and normal mice at the time of the inoculation (Table 2A). The cells from control animals showed a decline of CFU-s for 3 weeks, followed by a marked increase through 8 weeks, a pattern usually seen in this culture system. In contrast there were fewer CFU-s at the start and no recovery in cells from exposed animals. The number of nonadherent cells in the cells from the suspensions was similar for both groups; however, the number of CFU-s in both groups was quite different during the culture period (Table 2B). The numbers of CFU-s drastically decreased after 5 weeks in the cultures formed from benzene-treated mice, approaching near zero levels by 10 weeks of the culture, whereas CFU-s numbers were maintained for 13 weeks in the cultures formed from normal mice (Tables 2A and B). Granulocytes were not affected by exposure to benzene, nor were monocytes and macrophages until 10 weeks (Table 3). This shift to macrophages and monocytes usually indicates a senescent culture. During the culture period fewer
350
HARIGAYA TABLE
ET AL. SA
NUMBER OF SPLEEN COLONIES IN RECIPIENT MICE Time after reinoculation 0 3 Days 1 Week 4 Weeks 6 Weeks 10 Weeks
(NTN)’ 16.4 8.3 4.0 4.5 3.9 22.2
k + + + f *
1.0” 1.4 o.9b 0.6 o.9b l.5b
(NHB) 5.9 1.6 3.4 5.7 3.9 2.8
Note. 10’ nucleated cells inoculated. Mean ’ N, Normal control hone marrow cells; B, days (6 hr/day); B’, hone marrow cells from mice were maintained in room air for 5 days bn = 12.
+ + * + f r
o.9b 0.4 0.76 o.9b 0.6’ 0.4
III W-N) 16.4 9.1 4.4 8.6 15.2 0.2
+ + + f + +
(B’YB’)
(I& l.ob 1.3 1.3b l.Ob 1.5 0.26
5.9 2.3 3.9 4.7 5.6 6.2
+ f + + f +
o.9b 0.5 0.8” 0.7 0.9 0.7
12.5 5.4 5.8 8.3 20.7 0.4
-+ it -+ f k f
2.1b 1.0 1.2’ 0.9 1.0 0.3
? SEM, n = 15. hone marrow cells from mice exposed to benzene for 11 consecutive mice exposed to benzene for 9 days (5 days/week; 6 hr/day). The after the cessation of benzene exposure.
than 14% of the nonadherent cells from both groups were immature cells as shown in Table 3. Since the cultures formed from benzene-treated mice did not produce sufficient numbers of cells in suspension to inoculate into neonatal mice after 5 weeks of culture, only CFU-s measurements were performed thereafter. Feeding of these cultures was continued to 16 weeks of culture; no cellular transformation was observed in vitro. Neonatal mice were injected with bone marrow cell suspensions obtained at the time the primary cultures were established and with cell suspensions prepared from the nonadherent cells at the time the cultures were reinoculated and at 1, 3, and 5 weeks after reinoculation. The total number of neonates injected was 222; 108 received cells from benzene-exposed mice and 114 received cells from sham exposed mice. The mice were followed for 8 months. They gained weight normally and maintained normal white blood cell counts, differential cell counts, and hematocrits. The objective of the next experiment was to confirm the aforementioned observations and to examine whether the observed diminution of CFU-s in the cultures from benzene-treated mice was because of an injury to cells in the adherent cell layer or because of a direct injury of the CFU-s leading to
diminished self-replication. In addition, recovery from the benzene-induced injury was studied by commencing cultures 5 days after the cessation of benzene exposure. Five different combinations of adherent cell layers and reinoculated bone marrow cells were used as shown in Table 4. In the first four groups, adherent cell layers from control and exposed animals were reinoculated with cells from either control or exposed mice. The reduced frequency of CFU-s in the marrow of benzene-exposed mice required that a greater number of cells be reinoculated into these cultures. In the first group the number of reinoculated cells (4.5 X 106/flask) was adjusted as closely as possible to contain a similar number of CFU-s as in the cultures reinoculated with bone marrow cells from benzene-treated mice (the reduction of bone marrow cellularity was 35% in the benzenetreated group compared to the control group). The number of cells reinoculated into all cultures containing either adherent or nonadherent cell populations from benzene-exposed mice was 1 X 10’. The donor animals in groups 1 to 4 were exposed to either benzene or normal air for 11 consecutive days. In the fifth group, the donor animals were maintained in room air for 5 days after the cessation of the benzene exposure. The cultures containing adherent cells
HEMATOTOXICITY TABLE TOTAL
NUMBER
OF
Time after reinoculation 0
3 Days 1 Week 4 Weeks 6 Weeks 10 Weeks 12 Weeks
(N!N) 138 215.8 73.4 41.1 15.7 66.8 14.2
(29.2) (9.9) (5.6) (2.1) (9.1)
59
CFU-s PER FLASK IN DIFFERENT LAYERS
592 86.8 99.2 88.5 37.1 11.5 3.8
COMBINATIONS
(14.7) (16.8) (14.9) (6.3) (1.9)
OF ADHERENT
III (B-N) 1640 392.0 84.0 73.7 114.5 <1 1.6
CELL
CELLS
AND REINOCULATED
(NYB)
351
OF BENZENE
(23.9) (5.1) (4.5) (7.0)
(B’YB’) 592 116.5 90.2 96.5 87.8 25.1 2.4
(19.7) (15.2) (15.8) (14.8) (4.2)
1250 211.8 67.3 108.0 134.8
(16.9) (5.4) (8.6) (10.8)
a Percentage of initial inoculum of CFUs, eight flasks in each point. * Examination of adherent cell layers.
from normal bone marrow and reinoculated with normal bone marrow maintained CFUs for a longer period of time than any of the combinations (Tables 5A and B). The mean number of spleen colonies in mice receiving 1 X lo5 cells from lo-week cultures of normal adherent and reinoculated cells 7 days after the iv inoculation was 22.2 f 1.5. When cell suspensions from other combinations were assayed for CFU-S the numbers found were significantly lower. After 12 weeks of culture all of the supernatant fluid was removed. The adherent cells were washed twice with Dulbecco’s phosphate-buffered saline, pH 7.3, in which there was no Ca*+ orMg *+. Trypsin-EDTA (GIBCO) was used once to suspend to the adherent cells and then growth medium was added to terminate the reaction. All adherent cells from similarly prepared flasks were pooled, washed, and resuspended in Hepes-buffered MEM and assayed for CFU-s. The contents of CFU-s in the adherent cell layers of different groups were scant at this time, but the adherent cell layers in the first group (normal adherent-normal reinoculation) had higher numbers of CFU-s than the other groups (Table 5B). The endogenous colonies were always less than 0.2 +- 0.1 (mean + SEM, n = 15) per spleen in all CFU-s assays.
DISCUSSION The relationship between chronic benzene exposure and myelotoxicity has been recognized for over 80 years. The mechanism by which benzene induces marrow failure is unclear. It was believed that the cells most sensitive to benzene were those in early stages of development (Snyder et al., 1977; Uyeki et al., 1977). Recent reports show that benzene exposure induced a marked reduction of bone marrow stem cells and granulocyte-macrophage progenitors (Uyeki et al., 1977; Gill et al., 1980). Our data clearly demonstrate that benzene injures stem cells and alters their cellular environment leading to reduced stem cell replication in culture. The data further show that benzene injures cells that produce the adherent layer since adherent cell layers from benzene-exposed mice do not support the growth of stem cells in supernatant fluid derived from normal bone marrow as long as adherent cell layers derived from normal bone marrow support such growth. The seeding efficiency of cycling CFU-s is consistently lower than that observed for normal marrow in spleen colony assay (Monette and De Mello, 1979). During benzene inhalation (400 ppm/6 hr/day), the proportion of stem cells in cycle is increased
352
HARIGAYA
(Cronkite, unpublished). Accordingly, the numbers of CFU-s in marrow from benzeneexposed mice might be higher than the ones measured. If cycling CFU-s have an intrinsically lower capacity for self-renewal the more rapid depletion of CFU-s in cultures might be explained on this basis. It is unlikely that this could be the sole explanation since normal bone marrow grown with adherent cells from benzene-treated mice has lesser self-replication. The liquid culture system of murine bone marrow cells has recently been developed and its application in studies of chemical or viral leukemogenesis has been reported (Dexter and Lajtha, 1976; Dexter et al., 1977a; Teich and Dexter, 1978; Dexter and Teich, 1979; Greenberger, 1979). Ionizing radiation, chemical carcinogens, and viral carcinogens are known initiating agents that can induce long-lived preleukemic cells. The development of overt leukemia depends on additional changes that occur during the prolonged latency of the preleukemic cells in their host (Klein, 1979). In studies on leukemogenesis using donor mice treated with methylnitrosourea (MNU), Dexter and Teich (1979) cultured cells for 13 to 22 weeks and observed increased numbers of blast cells. These cultured cells produced leukemia when injected into syngeneic hosts. Our test system is based on the preceding observations and Gross’s ( 1970) demonstration that leukemic cells or virus produce leukemia in a few days to a few weeks after they are injected into neonatal mice. Hence if benzene were an initiator comparable to an alkylating agent as MNU, one would expect that in vivo exposure of mice to benzene would produce the initiating event and that the in vitro culture might allow a preleukemic or leukemogenic clone to emerge that could be detected by injection into neonatal mice. Our failure to observe leukemia suggests, but does not prove, that benzene is not an initiator of leukemia in mice. Lajtha (1979) believes that a long Go is needed for “rapair of DNA (genetic house-
ET AL.
keeping).” Thus the role of benzene may be more of a promoter by forcing proliferation of the hematopoietic pluripotent stem cell to maintain life saving hemopoiesis and thus accelerate the emergence of preleukemic and leukemogenic clones from stem cells that have been exposed to leukemogenic initiating agents prior to benzene exposure.
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(1981)
The Detection of in Wvo Hematotoxicity of Benzene by in vitro Liquid Bone Marrow Cultures’ KENICHI
HARIGAYA,*
MARILYN, E. MILLER, EUGENE AND ROBERT T. DREW
Medical Research Center, Brookhaven National
Laboratory,
P. CRONKITE,’
Upton, New York 11973
Received January 27, 1981: accepted April 24, 1981 The Detection of in Vivo Hematotoxicity of Benzene by in Vitro Liquid Bone Marrow Culture. HARIGAYA,K.,MILLER, M.E., CRONKITE,E. P., ANDDREW,R.T.(I~~I). Toxicol. Appl. Pharmacol. 60, 346-353. Bone marrow toxicity induced by benzene inhalation was investigated in mice using an in vitro bone marrow culture system and a bioassay for ‘leukemia. Donor animals (6-week-old female C57Bl/6J) were exposed to 400 ppm benzene 6 hr/day for either 9 days (5 days/week) or for I I consecutive days. Cell suspensions from long-term bone marrow cultures established from benzene-exposed mice showed a progressive reduction in the number of spleen colony-forming cells (the hematopoietic stem cell) compared to control. Different combinations of adherent cell layers and reinoculated cells showed that cultures containing bone marrow cells from benzene-exposed mice had a lower capacity to maintain stem cell proliferation than normal combinations irrespective of whether benzene-exposed marrow was in the adherent cell layers or reinoculated cells. These results indicate that benzene inhalation produces stem cell injury leading to diminished self-replication and derangement of the adherent marrow population. Bone marrow cultures from benzene-treated mice did not develop detectable transformation in vitro throughout the culture period. Neonatal mice inoculated with cultured cells from benzene-exposed mice have not developed leukemia during an S-month period after inoculation.
A continuous liquid culture system of bone marrow has facilitated the study of the proliferation and differentiation of hematopoietic stem cells and their cellular environment (Dexter et al., 1977b). In this mouse in vitro system, hematopoietic cell lineages consisting of granulocytes, monocytes, macrophages, megakaryocytes and erythrocytic and lymphocytic precursors are generated (Dexter et al., 1977b; Dexter, 1978; Testa and Dexter, 1978; Williams et al., 1978b; ’ Research supported by the Environmental Protection Agency under Agreement EPA-79-D-X0533, and the U.S. Department of Energy under Contract DEAC02-76CHOOO 16. 2 Present address: Department of Pathology, Keio University School of Medicine, Tokyo, Japan. 3 To whom reprint requests should be addressed 0041-008X/81/1
10346-08%02.00/O
Copyright 0 1981 by Academic Press. Inc. All rights of rcproduclion in any form reserved.
Johes-Villeneuve et al., 1980). There are two basic components in this culture system. The first is a cell population which adheres to the culture flask and is composed of phagocytic mononuclear cells, fat cells, endothelial cells, and epithelial cells. The second is a cell pop ulation suspended in the culture medium which when examined by light microscopy is found to contain mainly granulocytes and macrophages. The latter cell population is conveniently referred to as the nonadherent cell population. Spleen colony-forming cells (CFU-s, hematopoietic stem cells), are routinely maintained for at least 12 weeks using mouse bone marrow in this culture system (Dexter et al., 1977b). Some CFU-s in the nonadherent cell population arise from the adherent cell layers during the culture period 346
HEMATOTOXICITY TABLE MEASURED
CONCENTRATION EXP~WRE
1 OF BENZENE
IN THE
CHAMBERS
Expt groups”
Days of exposure
Benzene average concentration (mm * SW
Range of daily averages (ppm)
IP
9
411 f 20
392-446
IR
9
403 * 25
384-461
HP
11 9
376 -+ 21 392 + 13
348-408 374-408
IIR
11 9
410 +- 19 396 2 I
382-443 387-410
a I, Experiment I; II, Experiment II; P, benzene exposure of donor animals for the establishment of adherent cell layers; R, benzene exposure of donor animals for the reinoculated cells.
(Mauch et al., 1980). The spontaneous decline in stem cell replication appears to be attributed to the environment of the adherent portion of the marrow culture rather than to exhaustion of self-renewal potential in this bone marrow culture system (Dexter et al., 1977b; Williams et al., 1978a). The close relationship between chronic high-level benzene exposure and the occurrence of leukemia in humans has long been recognized (Brief et al., 1980, Snyder et al., 1977). Cronkite ( I96 1) reviewed benzene toxicity of man and concluded that any agent which produces marrow aplasia is a putative leukemogen. There have been reports of benzene-induced leukemia in mice (Lignac, 1932; Maltoni and Scarnato, 1979; Snyder et al., 1980). However, other investigators failed to demonstrate a relationship between exposure to benzene and the subsequent development of leukemia in mice (Thorpe, 1974; Ward et al., 1975). At the present time benzene exposure of man is associated with the development of leukemia but whether benzene is an initiator or promotor is not known. Benzene is unquestionably a myelotoxic substance which affects undifferentiated and more.differentiated-hematopoietic cells in mice (Lee et al., 1974;
347
OF BENZENE
Snyder et al., 1977; Uyeki et al., 1977; Gill et al., 1980). In this study, the mechanisms of myelotoxicity and leukemogenicity were investigated in mice after acute benzene inhalation by using a liquid culture system of bone marrow and a bioassay for leukemia (Gross, 1970). Our studies indicate that both the injury of hematopoietic stem cells and the derangement of their cellular environment are important in the myelotoxicity of benzene. Leukemogenicity has not been demonstrated 8 months after the inoculation of neonatal mice with cultured cells from benzene-exposed mice. METHODS Benzene exposure. Exposures to benzene were carried out in an isolation chamber system similar to that described by Laskin et al. (1970). Animals were housed in nesting boxes in the living quarters of the chamber and were allowed food and water ab libirum, except during the actual exposure periods. The room was maintained on a 12-hr light cycle. During exposure animals were placed in wire cages and then into a 128-liter exposure chamber mounted inside the isolation system. Control animals were housed in similar cages for exposure to air only. The chamber was operated at approximately 15 air changes/hr. Benzene vapors were generated by metering air through a bubbler and then into the airstream of the inhalation chamber. Concentrations were monitored at halfhour intervals using an automatic gas sampling coupled to a gas sampling loop in a Packard Model 417 gas chromatograph. The chroTABLE MEAN
Weeks after reinoculation 0 1 3 5 8 10 13
No.
2A
OF SPLEEN
COLONIES
IN RECIPIENT
MICE
Control 18.3 f 2.2 4.0 + 0.8 4.3 _+ 0.6 16.6 I! 2.1 Confluent’ 8.1 f 1.0 12.9 -+ 1.8
(CFU-s)
Benzene 7.6 2.9 3.3 1.6 1.3 0.4 0.2
f + 2 * + + f
1.2 0.5 0.6 0.3 0.4 0.3 0.2
Note. 10’ nucleated cells inoculated. Mean + SEM, n= IS. ’ Colonies too numerous to count (>30).
HARIGAYA
348 TABLE
and the contents of one femur and tibia were each flushed into a screw-capped. 25-cm* plastic culture flask (Corning Co., Corning, N.Y.) (one mouse yielding material for two flasks) containing Fischer’s culture medium (Grand Island Biological Co., Grand Island, N.Y.), supplemented with 25% horse serum (Flow Lab. Inc., McLean, Va.), antibiotics (100 U/ml penicillin and 100 pg/ml streptomycin), and IO-’ M hydrocortisone sodium succinate (Upjohn Co., Kalamazoo, Mich.). No attempt was made to obtain a single-cell suspension. The cultures were gassed with a mixture of 5% CO* in air, incubated at 33’C, and were fed once or twice a week by removal of half the growth medium and addition of an equal volume of fresh growth medium. After 3 weeks, all nonadherent cells were removed by decanting and the adherent cell layers were washed twice with Fischer’s medium. At this time a second group of 24 benzene-exposed mice was killed, the femurs and tibias were removed, pooled, and ground with a mortar and pestle. Single-cell suspensions were prepared by passing the suspension through successively finer gauge needles (18-27). and the cell concentration was determined by a Coulter electronic particle counter. Ten million freshly isolated cells were added in fresh medium to the adherent cell layer. This method of cell preparation was used for reinoculation in order to increase the cell yield, as well as to shorten the preparation time. Feeding of the cultures continued once or twice a week by removing half the growth medium and adding an equal volume of fresh growth medium. The suspensions removed from each flask were pooled, centrifuged at 8OOg for IO min. and then washed twice in Hepes-buffered minimal essential medium (MEM) (GIBCO). At appropriate time intervals, CFU-s and leukemogenicity in neonatal mice were determined using the pooled cell suspensions which were kept on ice during processing. Final cell suspensions were adjusted to appropriate cell numbers for CFU-s assays (I X 106/ml) and neonatal injection (2 X lO’/ml).
2B
TOTAL NUMBER OF CFU-S AND NONADHERENT CELLS IN SUSPENSION PER FLASK IN THE BONE MARROW CULTURES No. of nonadherent cells/flask (X IO’)
No. of CFU-s/f?ask Weeks after reinoculalion 0 I 3 5 a IO I3
COllld 1833 105 194.9 199.2 >480 48.9 38.7
BenZCne
(5.5) (10.6) (10.9) (226.2) (2.7) (2.1)
760 70.3 149.9 25.6 6.6 I .4 Cl
(9.9) (19.7) (3.4) (0.9) (0.2)
CO~l~Ol
Benzene
IW 2s 45 12 I6 6 3
100 24 45 I6 5 3 3
ET AL.
’ Percentage of initial inoculum of CFU-s. 34 flasks in each point.
matographic data were confirmed by wet chemical analysis for benzene vapors in the chamber using a bubbler with ethanol as the absorbing solution. The absorbance was measured at a wavelength of 254.6 nm on a uv spectrophotometer. Groups of 25 C57Bl/6J female mice (BNL) were exposed to either normal air or 400 ppm of benzene in chambers for either 9 days (6 hr/ day, 5 days per week) or 11 consecutive days (6 hr/day). The nominal concentration of benzene throughout these experiments was 400 ppm. The average measured concentration for any one experiment did not vary from that by more than 6%. The highest and lowest measured daily averages were 461 and 346 ppm, respectively (Table 1). Bone marrow cultures. On the day following the last exposure, mice were killed by cervical dislocation and dipped in 70% alcohol. Whole legs were removed, freed from muscle and kept in sterile plastic plates on ice until processed. One end of the bone was cut with scissors, TABLE
3
DIFFERENTIALS (%) OF CULTURED CELLS IN SUSPENSIONS Monocytes and macrophages
Granulocytes
Others”
Immature cells
Weeks after reinoculation
Control
Benzene
Control
Benzene
Control
Benzene
Control
Benzene
I 3 5 8 IO 16
66.5 64.0 26.0 1.0 39.0 75.0
73.0 58.5 29.0 3.5 3.5 0
17.5 33.5 72.0 98.5 44.5 18.0
13.5 39.0 69.5 96.5 96.5 100.0
14.0 2.5 1.5 0.5 13.5 7.0
13.5 2.0 I.5 0 0 0
0 0 0 0 3 0
0 0.5 0.5 0 0 0
’ Megakaryocytes or undetermined cells.
HEMATOTOXICITY TABLE FIVE DIFFERENT CELL LAYERS AND
4
COMBINATIONS REINOCULATED
OF ADHERENT BONE MARROW
CELLS
1. II. III. IV. V.
No. of flasks
Adherent cell layers
8 8 8 8 8
N N B B 8’
Reinoculation” (No. of cells to reinoculate) N (0.45 B (1 x N (1 X B (1 x B’ (1 x
x 10’) 10’) 10’) 10’) 107)
a N, Normal control bone marrow cells; B, bone marrow cells from mice exposed to benzene for 11 consecutive days (6 hr/day); B’, bone marrow cells from mice exposed to benzene for 9 days (5 days/week, 6 hr/day). Donor animals were maintained in room air for 5 days after the cessation of benzene exposure. Spleen colony assay. The spleen colony assay (Till and McCulloch, 1961) was performed in female C57BL/ 65 mice 8 to 12 weeks old. Groups of 12 to 15 mice were injected iv with 1 X IO5 cells 3 hr after 730 rad of whole body irradiation administered by a 250 kVp General Electric Maxitron X-ray machine operating at 250 kVp and 30 mA (target distance, 60 cm; 0.5 mm copper plus 1.0 mm aluminum filter; dose rate, 103-108 rad/min). The endogenous colonies were determined in animals receiving only Hepes-buffered MEM. Spleens were harvested 7 days later, and colonies were counted at 3X magnification after at least 24 hr fixation in Bouin’s solution. The endogenous spleen colony count was subtracted from the spleen colony counts obtained from mice injected with bone marrow cells. Leukemogenecity in neonotal mice. Newborn (less than 48 hr old) C57B1/6J mice, inoculated ip with 5 X IO5 cultured cells from benzene-exposed or control mice, were weighed and evaluated for splenomegaly and death weekly after weaning. Microhematocrits and morphological examination of peripheral blood were performed monthly.
RESULTS On the day following the last exposure, the femur and tibia from a single mouse were flushed into an individual culture flask to establish the adherent cell layer (40 flasks). Single cell suspensions were prepared by flushing femurs and tibias from four sepa-
OF BENZENE
349
rate mice and immediately testing for CFUs content and leukemogenic potential in neonatal mice. After 3 weeks, a second cohort of mice treated identically were killed and single-cell suspensions of bone marrow were prepared by grinding marrow with a mortar and pestle. The single-cell suspensions were assayed for CFU-s content and leukemogenicity. An aliquot of the pooled, single-cell suspension was added to each of the established bone marrow monolayer cultures (1 X lo7 nucleated cells/flask). There were no morphologic differences between the adherent cell layers formed from benzene-treated and normal mice when examined with the inverted microscope. The culture suspensions were examined for CFUs and leukemogenicity at appropriate times during the culture period. There was a 30% reduction in the cell counts of bone marrow cell suspensions from benzene-treated animals. There were marked differences in the CFU-s frequencies between bone marrow cells from benzene-exposed and normal mice at the time of the inoculation (Table 2A). The cells from control animals showed a decline of CFU-s for 3 weeks, followed by a marked increase through 8 weeks, a pattern usually seen in this culture system. In contrast there were fewer CFU-s at the start and no recovery in cells from exposed animals. The number of nonadherent cells in the cells from the suspensions was similar for both groups; however, the number of CFU-s in both groups was quite different during the culture period (Table 2B). The numbers of CFU-s drastically decreased after 5 weeks in the cultures formed from benzene-treated mice, approaching near zero levels by 10 weeks of the culture, whereas CFU-s numbers were maintained for 13 weeks in the cultures formed from normal mice (Tables 2A and B). Granulocytes were not affected by exposure to benzene, nor were monocytes and macrophages until 10 weeks (Table 3). This shift to macrophages and monocytes usually indicates a senescent culture. During the culture period fewer
350
HARIGAYA TABLE
ET AL. SA
NUMBER OF SPLEEN COLONIES IN RECIPIENT MICE Time after reinoculation 0 3 Days 1 Week 4 Weeks 6 Weeks 10 Weeks
(NTN)’ 16.4 8.3 4.0 4.5 3.9 22.2
k + + + f *
1.0” 1.4 o.9b 0.6 o.9b l.5b
(NHB) 5.9 1.6 3.4 5.7 3.9 2.8
Note. 10’ nucleated cells inoculated. Mean ’ N, Normal control hone marrow cells; B, days (6 hr/day); B’, hone marrow cells from mice were maintained in room air for 5 days bn = 12.
+ + * + f r
o.9b 0.4 0.76 o.9b 0.6’ 0.4
III W-N) 16.4 9.1 4.4 8.6 15.2 0.2
+ + + f + +
(B’YB’)
(I& l.ob 1.3 1.3b l.Ob 1.5 0.26
5.9 2.3 3.9 4.7 5.6 6.2
+ f + + f +
o.9b 0.5 0.8” 0.7 0.9 0.7
12.5 5.4 5.8 8.3 20.7 0.4
-+ it -+ f k f
2.1b 1.0 1.2’ 0.9 1.0 0.3
? SEM, n = 15. hone marrow cells from mice exposed to benzene for 11 consecutive mice exposed to benzene for 9 days (5 days/week; 6 hr/day). The after the cessation of benzene exposure.
than 14% of the nonadherent cells from both groups were immature cells as shown in Table 3. Since the cultures formed from benzene-treated mice did not produce sufficient numbers of cells in suspension to inoculate into neonatal mice after 5 weeks of culture, only CFU-s measurements were performed thereafter. Feeding of these cultures was continued to 16 weeks of culture; no cellular transformation was observed in vitro. Neonatal mice were injected with bone marrow cell suspensions obtained at the time the primary cultures were established and with cell suspensions prepared from the nonadherent cells at the time the cultures were reinoculated and at 1, 3, and 5 weeks after reinoculation. The total number of neonates injected was 222; 108 received cells from benzene-exposed mice and 114 received cells from sham exposed mice. The mice were followed for 8 months. They gained weight normally and maintained normal white blood cell counts, differential cell counts, and hematocrits. The objective of the next experiment was to confirm the aforementioned observations and to examine whether the observed diminution of CFU-s in the cultures from benzene-treated mice was because of an injury to cells in the adherent cell layer or because of a direct injury of the CFU-s leading to
diminished self-replication. In addition, recovery from the benzene-induced injury was studied by commencing cultures 5 days after the cessation of benzene exposure. Five different combinations of adherent cell layers and reinoculated bone marrow cells were used as shown in Table 4. In the first four groups, adherent cell layers from control and exposed animals were reinoculated with cells from either control or exposed mice. The reduced frequency of CFU-s in the marrow of benzene-exposed mice required that a greater number of cells be reinoculated into these cultures. In the first group the number of reinoculated cells (4.5 X 106/flask) was adjusted as closely as possible to contain a similar number of CFU-s as in the cultures reinoculated with bone marrow cells from benzene-treated mice (the reduction of bone marrow cellularity was 35% in the benzenetreated group compared to the control group). The number of cells reinoculated into all cultures containing either adherent or nonadherent cell populations from benzene-exposed mice was 1 X 10’. The donor animals in groups 1 to 4 were exposed to either benzene or normal air for 11 consecutive days. In the fifth group, the donor animals were maintained in room air for 5 days after the cessation of the benzene exposure. The cultures containing adherent cells
HEMATOTOXICITY TABLE TOTAL
NUMBER
OF
Time after reinoculation 0
3 Days 1 Week 4 Weeks 6 Weeks 10 Weeks 12 Weeks
(N!N) 138 215.8 73.4 41.1 15.7 66.8 14.2
(29.2) (9.9) (5.6) (2.1) (9.1)
59
CFU-s PER FLASK IN DIFFERENT LAYERS
592 86.8 99.2 88.5 37.1 11.5 3.8
COMBINATIONS
(14.7) (16.8) (14.9) (6.3) (1.9)
OF ADHERENT
III (B-N) 1640 392.0 84.0 73.7 114.5 <1 1.6
CELL
CELLS
AND REINOCULATED
(NYB)
351
OF BENZENE
(23.9) (5.1) (4.5) (7.0)
(B’YB’) 592 116.5 90.2 96.5 87.8 25.1 2.4
(19.7) (15.2) (15.8) (14.8) (4.2)
1250 211.8 67.3 108.0 134.8
(16.9) (5.4) (8.6) (10.8)
a Percentage of initial inoculum of CFUs, eight flasks in each point. * Examination of adherent cell layers.
from normal bone marrow and reinoculated with normal bone marrow maintained CFUs for a longer period of time than any of the combinations (Tables 5A and B). The mean number of spleen colonies in mice receiving 1 X lo5 cells from lo-week cultures of normal adherent and reinoculated cells 7 days after the iv inoculation was 22.2 f 1.5. When cell suspensions from other combinations were assayed for CFU-S the numbers found were significantly lower. After 12 weeks of culture all of the supernatant fluid was removed. The adherent cells were washed twice with Dulbecco’s phosphate-buffered saline, pH 7.3, in which there was no Ca*+ orMg *+. Trypsin-EDTA (GIBCO) was used once to suspend to the adherent cells and then growth medium was added to terminate the reaction. All adherent cells from similarly prepared flasks were pooled, washed, and resuspended in Hepes-buffered MEM and assayed for CFU-s. The contents of CFU-s in the adherent cell layers of different groups were scant at this time, but the adherent cell layers in the first group (normal adherent-normal reinoculation) had higher numbers of CFU-s than the other groups (Table 5B). The endogenous colonies were always less than 0.2 +- 0.1 (mean + SEM, n = 15) per spleen in all CFU-s assays.
DISCUSSION The relationship between chronic benzene exposure and myelotoxicity has been recognized for over 80 years. The mechanism by which benzene induces marrow failure is unclear. It was believed that the cells most sensitive to benzene were those in early stages of development (Snyder et al., 1977; Uyeki et al., 1977). Recent reports show that benzene exposure induced a marked reduction of bone marrow stem cells and granulocyte-macrophage progenitors (Uyeki et al., 1977; Gill et al., 1980). Our data clearly demonstrate that benzene injures stem cells and alters their cellular environment leading to reduced stem cell replication in culture. The data further show that benzene injures cells that produce the adherent layer since adherent cell layers from benzene-exposed mice do not support the growth of stem cells in supernatant fluid derived from normal bone marrow as long as adherent cell layers derived from normal bone marrow support such growth. The seeding efficiency of cycling CFU-s is consistently lower than that observed for normal marrow in spleen colony assay (Monette and De Mello, 1979). During benzene inhalation (400 ppm/6 hr/day), the proportion of stem cells in cycle is increased
352
HARIGAYA
(Cronkite, unpublished). Accordingly, the numbers of CFU-s in marrow from benzeneexposed mice might be higher than the ones measured. If cycling CFU-s have an intrinsically lower capacity for self-renewal the more rapid depletion of CFU-s in cultures might be explained on this basis. It is unlikely that this could be the sole explanation since normal bone marrow grown with adherent cells from benzene-treated mice has lesser self-replication. The liquid culture system of murine bone marrow cells has recently been developed and its application in studies of chemical or viral leukemogenesis has been reported (Dexter and Lajtha, 1976; Dexter et al., 1977a; Teich and Dexter, 1978; Dexter and Teich, 1979; Greenberger, 1979). Ionizing radiation, chemical carcinogens, and viral carcinogens are known initiating agents that can induce long-lived preleukemic cells. The development of overt leukemia depends on additional changes that occur during the prolonged latency of the preleukemic cells in their host (Klein, 1979). In studies on leukemogenesis using donor mice treated with methylnitrosourea (MNU), Dexter and Teich (1979) cultured cells for 13 to 22 weeks and observed increased numbers of blast cells. These cultured cells produced leukemia when injected into syngeneic hosts. Our test system is based on the preceding observations and Gross’s ( 1970) demonstration that leukemic cells or virus produce leukemia in a few days to a few weeks after they are injected into neonatal mice. Hence if benzene were an initiator comparable to an alkylating agent as MNU, one would expect that in vivo exposure of mice to benzene would produce the initiating event and that the in vitro culture might allow a preleukemic or leukemogenic clone to emerge that could be detected by injection into neonatal mice. Our failure to observe leukemia suggests, but does not prove, that benzene is not an initiator of leukemia in mice. Lajtha (1979) believes that a long Go is needed for “rapair of DNA (genetic house-
ET AL.
keeping).” Thus the role of benzene may be more of a promoter by forcing proliferation of the hematopoietic pluripotent stem cell to maintain life saving hemopoiesis and thus accelerate the emergence of preleukemic and leukemogenic clones from stem cells that have been exposed to leukemogenic initiating agents prior to benzene exposure.
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