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As can be seen in Figure 1, ip treatment of mice with benzene results in an overall depression of bone marrow cellularity selective for the lymphoid and erythroid lineages, while the granulocytic lineages actually show an increase in cell number and a shift toward granulocytic differentiation. These results directly support those previously obtained by Seidel (14), and Dempster and Snyder (15). A more in-depth examination of the effects of benzene (and its metabolite HQ) on the induction of granulocytic differentiation in mice (Figure 2) indicated that benzene was able to stimulate the differentiation of myeloblasts, as measured by an increase in the percent of promyelocytes and intermediate granulocytic progenitors in the bone marrow one day after the last benzene injection (3 days after the initiation of treatment), but had no effect on the number of myeloblasts. Neither was the number of mature granulocytes stimulated by day 3. However, in experiments carried out for 7 days, benzene significantly increased the number of mature granulocytes compared to control values (Figure 3). The experiments presented here are representative of three such experiments each, all of which gave similar results. Identical results were obtained when the data were expressed as percentage of total cells counted. Hydroquinone administered to mice for 3 days, at a dose of 50 mg/kg bw, also stimulated granulocytic differentiation as indicated by an increased percentage of promyelocytes and intermediate granulocytic progenitors (Figure 2). In contrast to benzene, HQ doubled the number of myeloblasts and stimulated differentiation to GB forms to a greater extent than benzene, yet the terminal differentiation of GB to mature granulocytes was limited. HQ administered at 25 mg/kg body weight caused a lesser, but significant, stimulation of granulopoiesis (data not shown). At these doses of benzene or HQ, there was no loss of animals, nor any overt signs of toxicity, and the viability of the cells flushed from the femurs was always >98%.
Figure 3. The induction of granulocytic differentiation in C57BL/6J mice by benzene over time. Groups of mice (n=4) were treated with benzene as described for Figures 1 and 2. Several groups were sacrificed, and their nucleated bone marrow cells harvested, 18 hr after the last benzene injection (3 days after the initiation of treatment) as before. However, several other groups were maintained for an additional 2 days and then sacrificed (5 days after treatment initiation), and still other groups were maintained for 2 more days before being sacrificed (7 days after treatment initiation). All total and differential counts were determined as for Figures 1 and 2. PM, promyelocytes; GB, myelocytes, metamyelocytes, and band(ring) forms; GS, mature segmented granulocytes.
Granulocytic Differentiation of Myeloblasts by Benzene and HydroquinoneThese in vivo results suggested that benzene per se, or by its metabolism to HQ, is capable of inducing granulocytic differentiation. However, these results do not indicate whether either compound causes induction directly or indirectly in vivo. To answer this question and to develop a system for use in further mechanistic studies involving the induction of granulocytic differentiation, we turned to the use of myeloid cell lines. The well-studied human promyelocytic leukemia cell line, HL-60 (19), which consists of approximately 35 to 45% myeloblasts and 55 to 65% promyelocytes, was selected because it has been used in many studies as a surrogate for the GM-CFU and because a number of agents have been shown to induce terminal granulocytic differentiation in these cells (19). The normal IL-3-dependent mouse myeloblastic 32D.3 (G) cell line was chosen because it has been adapted to terminally differentiate to granulocytes in response to G-CSF (18) and because any data obtained using this cell line can be directly compared with that obtained from the mouse.
Benzene-induced Granulocytic Differentiation in HL-60 CellsAs can be seen from the data presented in Table 1 and Figure 4, exposure of HL-60 myeloblasts to benzene, followed by incubation for 7 days, resulted in terminal granulocytic differentiation as measured by morphology (determination of the percent of cells classified as myelocytes or higher granulocytic forms) and the development of several characteristics of the granulocytic phenotype. No significant induction of nonspecific esterase activity (Figure 4), indicative of monocytes, was detected, nor was monocyte morphology observed (Table 1). The ability of benzene to induce granulocytic morphology, as well as the appearance of other markers of the granulocytic phenotype, was a function of the concentration of benzene used over the range of 0.1 mM to 5 mM (data not shown). As can be seen in Table 1, benzene treatment of HL-60 cells caused a significant decrease in the number of normally occurring myeloblasts and promyelocytes with a corresponding shift toward more intermediate progenitors and mature granulocytes. The majority of the differentiated progenitors appeared to be metamyelocytes and myelocytes, but a significant number of terminally differentiated granulocytes was observed (Table 1).
Involvement of Protein Kinase C and the 5-LPO Pathway in Benzene-induced Granulocytic Differentiation of HL-60 CellsSustained activation of protein kinase C (PKC) is essential to the induction of differentiation in HL-60 cells (20). Both retinoic acid and dimethylsulfoxide (DMSO) induce HL-60 cells to differentiate to granulocytes by activation of PKC (21). Benzene activates PKC in intact rabbit platelets (22) and in the macrophagelike cell lines, P388D1 and BAC1.2F5CSF-1 (Carlson and Kalf, unpublished data). An indication that benzene also activates PKC in HL-60 cells is provided by the benzene-induced phosphorylation of cellular proteins in these cells, as well as the ability of benzene-treated HL-60 cell membrane preparations to carry out the phosphorylation of histone III-S, a PKC substrate, in an in vitro assay (23,24). Sphinganine, a potent inhibitor of PKC in vitro and in cell systems (25), prevents not only benzene-induced phosphorylation of cellular proteins, but also differentiation to granulocytes (23,24), implicating the phosphorylation of these proteins by PKC in benzene-induced granulocytic differentiation of HL-60 cells. An active LPO pathway of metabolism that converts arachidonic acid (AA) to the peptidoleukotriene, LTD4, is essential for normal (26,27) as well as leukemic (28,29) myeloid progenitor cell differentiation. LTD4 is an essential intermediate in CSF-induced clonal growth of GM-CFU (26,27). It has been demonstrated that HL-60 cells express a granulocytic phenotype if, during induction, PKC-activated release of AA from plasma membranes, and lipoxygenation of AA to LTD4, occurs (26). Benzene-treatment of HL-60 cells results in a release of AA from membrane lipids and a subsequent increase in LTD4, both of which are inhibited by sphinganine (23,24).
Several inhibitors of 5-LPO were tested for their ability to prevent benzene-induced granulocytic differentiation of HL-60 cells as measured by morphology and a functional parameter, superoxide production (NBT reduction). From Table 2 it is apparent that the highly specific 5-LPO inhibitor, AA-861 (30), as well as caffeic acid, prevented benzene-induced granulocytic differentiation and that this inhibition could be overcome by the concomitant addition of LTD4 to the reaction. To further implicate LTD4 in the differentiation process, we tested the ability of LTD4 receptor antagonists to inhibit benzene-induced differentiation. HL-60 cells were incubated with benzene in the presence or absence of MK-571, a highly specific inhibitor of LTD4 binding to its high affinity receptor (31). Benzene-induced granulocytic differentiation of HL-60 cells was completely inhibited by the LTD4 receptor antagonist and differentiation was restored by the addition of excess LTD4 (Figure 5). Another receptor antagonist, LY163443 (32), gave similar results (data not shown). Taken together, these results indicate that both production of LTD4, and its interaction with, and activation of, its receptor are essential for benzene-induced granulocytic differentiation.
Granulocytic Differentiation of the Myeloblastic Cell Line 32D.3 (G) by Benzene and HydroquinoneTo compare the induction of granulocytic differentiation by benzene and HQ with that of the normal physiological inducer, G-CSF, we turned to the IL-3 dependent diploid myeloblastic cell line, 32D.3 (G), which was derived from normal mouse bone marrow and adapted to differentiate in the presence of G-CSF (18). Table 3 presents data from one representative of four experiments that gave similar results. Few differentiated myeloid cells were detected in the presence of IL-3 alone, which, although obligatory for proliferation and survival of the myeloblast, does not induce differentiation. rHuG-CSF, which is fully capable of binding to the mouse G-CSF receptor, induced terminal differentiation to granulocytes. Substitution of noncytotoxic concentrations of benzene or HQ in place of G-CSF also induced differentiation in 32D myeloblasts (Table 3). While both benzene and HQ replaced the necessity of G-CSF for differentiation to granulocytes, neither was able to obviate the dependence of the cells on IL-3 for survival and growth (data not shown).
Granulocytic Differentiation of 32D Myeloblasts by LTD4Since LTD4 is a downstream effector of G-CSF-induced signal transduction and benzene has been shown to produce LTD4 via activation of PKC with subsequent release of AA (23,24) and upregulation of the 5-LPO pathway (Table 2), we tested the ability of LTD4 to replace either G-CSF or benzene (as well as HQ) in the induction of granulocytic differentiation in 32D myeloblasts. As can be seen in Figure 6, LTD4 is capable of replacing these inducing agents in the concentration-dependent induction of terminal differentiation in 32D myeloblasts.
Effects of 5-LPO Inhibitors and LTD4 Receptor Antagonists on the Ability of Hydroquinone to Induce Granulocytic Differentiation in 32D Myeloblasts
The induction of differentiation by G-CSF (Table 4A) and benzene (data not shown) is inhibited by a specific 5-LPO inhibitor, as expected, since both G-CSF (33) and benzene induce the release of AA (the substrate for 5-LPO) from plasma membranes (23,24). However, the ability of HQ to induce differentiation is not prevented by the 5-LPO inhibitor, suggesting that HQ, in contrast to G-CSF and benzene, does not induce the formation of LTD4, but rather functions in some other way, perhaps by interacting with the LTD4 receptor. Therefore, we carried out an experiment to ascertain whether the ability of HQ to induce terminal granulocytic differentiation in 32D myeloblasts could be prevented by an LTD4 receptor antagonist. The addition of the specific antagonist MK-571 completely blocked HQ-induced terminal granulocytic differentiation (Table 4B) indicating that HQ may interact with the ligand-binding domain of the receptor to activate its signal and initiate the cascade of events that result in granulocytic differentiation. Hydroquinone can be oxidized in the cell to p-benzoquinone (BQ), a bifunctional alkylating agent that can interact indiscriminately with available sulfhydryl groups on membrane-bound proteins (4,34). Therefore, the possibility exists that BQ may bind covalently to sulfhydryl groups located at, or very near, the ligand-binding domain of the LTD4 receptor, obviating the need for LTD4 and activating the receptor in a constitutive, rather than signal-driven manner (as is the case with LTD4).
Effects of LTD4 versus HQ on 32D Myeloblast Proliferation and Granulocytic DifferentiationThe indication from LTD4 receptor antagonist studies that HQ, like LTD4, induces granulocytic differentiation through direct interaction with, and activation of, the LTD4 receptor was an interesting one. To further study this phenomenon, we examined the effects of both LTD4 and HQ on 32D cell proliferation and differentiation, in the presence and absence of IL-3, over time. As can be seen in Figure 7, neither LTD4 nor HQ can induce a signal for proliferation in 32D myeloblasts in the absence of IL-3. However, in the presence of IL-3, both LTD4 and HQ showed a significant stimulation of cell growth which, in the case of HQ, was more than 100% at day 4. LTD4 and HQ were both also capable of transmitting a signal for differentiation in the presence (data not shown) or absence of IL-3 (Figure 8). In the presence of the minimal concentration (3 U/ml) of IL-3 required for survival and proliferation of 32D myeloblasts, each inducer caused 100% total differentiation, but the degree of terminal differentiation was delayed in comparison with cells grown in the absence of IL-3 (data not shown). The proliferative signal provided by IL-3 competes with the differentiation signal from the inducer such that the degree of both total and terminal differentiation is decreased at 6 days.
An analysis of the kinetics of stage-specific granulocytic differentiation was performed on 32D myeloblasts induced by LTD4 or HQ in the absence of IL-3. This allowed for a determination of the inductive effects of LTD4, or HQ, in the absence of any competitive proliferative signal from IL-3. Figure 8 demonstrates that LTD4 was incapable of providing a proliferative signal, as evidenced by the low number of promyelocytes detected over the 6-day period, but was capable of producing a significant number of terminally differentiated granulocytes, as well as an increased number of intermediate progenitors. At each time point examined, the predominate cell type (differentiation stage) present was the mature segmented granulocyte. Absence of a proliferative burst was also detected upon HQ treatment; but, unlike LTD4, HQ induced predominately myelocytes at any given time point (Figure 8). HQ appears unable to induce significant differentiation beyond this point and, therefore, results in an incomplete differentiation program in these cells.
Exposure of C57BL/6J mice to a dose of benzene (35) known to depress other hematopoietic cell lineages (Figure 1) significantly stimulated granulocytic differentiation as well as the total number of granulocytes, indicating that granulopoiesis was also occuring (Figure 2). While benzene could not provide a growth signal for myeloblasts, it did stimulate their differentiation to promyelocytes and intermediate progenitors, but did not stimulate the production of mature granulocytes by day 3. However, when measured at day 7, benzene had increased the number of segmented mature granulocytes (Figure 3). Our results show a general increase in granulocytes after benzene exposure and are consistent with those that show an increase in the number of granulocytes in bone marrow of DBA/2J mice after short-term exposure to benzene (15). Hydroquinone, a major metabolite of benzene found in the bone marrow, also stimulated proliferation and differentiation of granulocytic progenitor cells by day 3 when administered to C57BL/6J mice (Figure 2) as well as in mouse 32D myeloblasts in culture (Figures 7, 8). In contrast to benzene, HQ provided both proliferative and differentiative signals for myeloblasts in vivo that increased the numbers of all progenitor forms, but appeared incapable of inducing terminal differentiation (Figure 2) for reasons that are not yet clear. To study the mechanism(s) whereby benzene and HQ stimulate granulopoiesis, we investigated whether the inductive effect of benzene or HQ on granulocytic differentiation could be reproduced in myeloblasts in culture. Benzene at noncytotoxic concentrations caused a dose-dependent specific induction of terminal granulocytic differentiation in HL-60 myeloblasts (Table 1; Figure 4), as measured by morphological and functional parameters (superoxide production, chloroacetate esterase activity, and the appearance of the specific surface antigen L-12-2) that are specific for granulocytic differentiation. Benzene induction produced a majority of intermediate progenitors (myelocytes, metamyelocytes and band forms); however, a significant number of mature granulocytes were also observed (Table 1). These results correspond to earlier observations that the granulocytic differentiation of HL-60 cells induced by certain agents is somewhat incomplete and defective (19). As was previously shown, activation of PKC appears to be involved in the induction of granulocytic differentiation in benzene-treated HL-60 myeloblasts. This is because the phosphorylation of cellular proteins, as well as the induced differentiation, was prevented by the concomitant presence of benzene and the specific PKC inhibitor sphinganine (23,24). Benzene-induced differentiation of HL-60 myeloblasts to granulocytes also appears to involve the induction of a functioning 5-LPO pathway for production of LTD4, since differentiation is inhibited by the 5-LPO inhibitors caffeic acid and AA-861 (Table 2) and by the specific LTD4 receptor antagonist, MK-571 (Figure 5). Inhibition by each of these agents was prevented by the concomitant addition of LTD4. These results together support the view that a role for benzene in the induction of granulocytic differentiation in HL-60 myeloblasts--like that of G-CSF--is to produce LTD4, which is a necessary signal for granulocytic differentiation. Although G-CSF has not yet been demonstrated to directly cause the production of LTD4, it has been shown to cause the activation of phospholipase A2 and the subsequent release of AA from cell membranes (33).
Benzene is also capable of inducing granulocytic differentiation in the diploid IL-3-dependent murine myeloblastic cell line, 32D.3 (G), whereas IL-3 induces only a growth response (Table 3). rHuG-CSF provides not only a differentiation signal (Table 3), but, in the absence of IL-3, a growth signal for 32D myeloblasts as well (24). Benzene, unlike G-CSF, is incapable of inducing growth in the absence of IL-3, but does provide the differentiation signal, and neither G-CSF nor benzene can synergize with IL-3 to stimulate growth (24). LTD4 and HQ are also incapable of supporting growth of 32D cells in the absence of IL-3, but can synergize with IL-3 to enhance growth (Figure 7). Both LTD4 and HQ induce granulocytic differentiation in 32D myeloblasts (Figure 8). Either benzene or HQ (Table 3), as well as LTD4 (Figure 6), can replace G-CSF for induction of differentiation. Note that, in the experiment reported in Table 3, no effort was made to optimize concentrations, so that the magnitude of benzene- or HQ-induced differentiation cannot be quantitatively compared with that of G-CSF. These results, in total, indicate that benzene, with its metabolite HQ, can induce granulopoiesis in 32D mouse myeloblasts since both the number of progenitor cells and terminally differentiated granulocytes increased; and they support our in vivo results concerning the induction of granulopoiesis by benzene and HQ in mice (Figure 2). The ability of HQ to synergize with other cytokines/growth factors (as seen in Figure 7 for IL-3) has been previously documented. Irons et al. (36), showed that pretreatment of nonadherent murine bone marrow cells, or lineage-restricted hematopoietic cells, with HQ, in vitro, followed by culture in complete medium for 8 days, significantly enhanced the number of granulocyte-macrophage colonies induced by recombinant GM-CSF. Optimal enhancement was observed with 1 The fact that benzene-induced granulocytic differentiation of myeloblasts in culture is prevented by inhibitors of PKC and 5-LPO supports the view that benzene-induced differentiation might also be due to benzene per se, not only the result of its metabolism to HQ in the myeloblast. Consequently, not all of the granulopoietic activity of benzene in vivo can be attributed to its metabolism to HQ. During chronic benzene exposure, benzene and HQ are both present in the bone marrow; therefore both can contribute to granulocytic differentiation. On the basis of results presented here and those previously obtained in our laboratory, benzene, like G-CSF, appears to activate the AA cascade and upregulate the 5-LPO pathway for the production of LTD4. Hydroquinone appears to obviate the requirement for LTD4 by activating the LTD4 receptor directly since specific LTD4 receptor antagonists, but not 5-LPO inhibitors, prevent HQ-induced granulocytic differentiation in myeloblasts. One can speculate about the roles of benzene and HQ in benzene-induced AML. As a known clastogen, HQ may cause a leukemogenic initiating event in the myeloblast [i.e., one of the translocations or deletions characteristic of AML (4)], and at the same time covalently bind to the LTD4 receptor to constitutively activate this signal-driven process and induce granulocytic differentiation, which is incomplete and arrested at the myelocyte stage. Blocks in the developmental program of terminal hematopoietic cell differentiation appear to be a major step in tumor progression (37). The ability of HQ to stimulate an increase in the number of myeloblasts, both in vivo (Figure 2) and in vitro (Figure 7) by an as yet undetermined mechanism, may then result in the classic picture of AML: an overproliferation of incompletely differentiated myeloid cells. Concomitantly, benzene may provide a promotional effect on the initiated myeloblast, via constitutive activation of PKC, and overexpression of its activity, with resultant pleotropic effects on morphology and growth control.
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M HQ in Dulbecco's phosphate-buffered saline minus Ca2+ and Mg2+ (PBS)/2 mM glucose [PBS-A] for 30 min at 37°C, after which the cells were collected, washed twice with PBS, and placed into culture in IMDM/2 mM glutamine/10% FBS with or without 3 U/ml rMuIL-3. LTD4 [as well as recombinant human G-CSF (rHuG-CSF)], the LPO inhibitors, and the LTD4 receptor antagonists were added as for HL-60 cells, but in the presence or absence of IL-3. Unless otherwise indicated (in figure or table legends), a sample of each culture was removed for cell counting after 3 days of incubation, and the remainder of the culture diluted to 2.5X105 cells/ml to ensure optimal growth conditions. Incubation was continued for 4 days, after which the cells were analyzed for the granulocytic phenotype.
Figure 1. Benzene-induced selective depression of bone marrow cellularity in C57BL/6J mice. Groups (n=4) of male C57BL/6J mice were established. One group served as the control and received only corn oil. The other groups were injected ip with benzene (600 mg/kg bw) in corn oil twice per day, 7 hr apart, for 2 days. Eighteen hours after the final injection, the animals were sacrificed by cervical dislocation, their femurs removed, and the nucleated bone marrow cells obtained and counted as described under methods. An analysis of the total numbers of cells of the various lineages in the bone marrow was carried out. A sample of the bone marrow cell suspension was used to prepare a slide for differential count. The slide was stained and analyzed as described under methods. For each slide 500 cells were counted and the percentage of each cell type determined. This percentage was multiplied by the total number of cells per femur to determine the number of a given cell type, and that was normalized to the number of cells in the control group. Data are expressed as the mean +/-SD (n=4). Where there were differences between groups, they were significant at the p</=0.01 level. Lymph, lymphocytes; NEC, nucleated erythroid cells; PM, promyelocytes; Neutr, neutrophils; Eos, eosinophils.
Figure 2. The induction of granulocytic differentiation in C57BL/6J mice by benzene or HQ. Groups of mice (n=4) were injected with benzene (600 mg/kg bw) in corn oil or HQ (50 mg/kg bw) in PBS-A, twice daily for 2 days. The control animals received corn oil or PBS-A only. Eighteen hours after the final injection, the mice were killed and their bone marrow obtained. A morphological analysis of the bone marrow was carried out as described under methods. Data are presented as absolute numbers of myeloid cells per femur represented by blast cells and total granulocytic cells of 500 cells analyzed. Qualitatively similar results were obtained when the data were expressed as the percent of nucleated bone marrow cells. The results are expressed as the mean +/-SD (n=4); *p</=0.001 as compared with the control group. The data for various cell types from the individual marrows are extremely tight (they range from SD +/- 0.001 to +/-0.03) and too small to be plotted by the printer/plotter; therefore, no error bars are visible. PM, promyelocytes; GB, myelocytes, metamyelocytes, and band (ring) forms; GS, segmented mature granulocytes.
Figure 4. Benzene-induced granulocytic differentiation of HL-60 myeloblasts. Cells (5X105/ml) were cultured with, and without, 5 mM benzene, as described in the methods section, for up to 7 days. The development of the granulocytic phenotype was determined by measuring those characteristics presented in the figure. Each analysis was carried out as described in the methods section; each set of values represents the mean +/-SD of three experiments, where each sample was carried out in triplicate. * Significantly different from the control cells at p</=0.001. Reprinted from Hazel et al. (24) with permission of AlphaMed Press.
Figure 5. Inhibition of benzene-induced granulocytic differentiation in HL-60 cells by an LTD4 receptor antagonist. Cells (5X105/ml) were preincubated with 1 
Figure 6. LTD4-induced granulocytic differentiation in 32D myeloblasts. Cells (2.5X105/ml) in IMDM (3 U/ml rMuIL-3; 10% FBS) were incubated with or without LTD4 for 3 days at 37°C, 5% CO2. Cells were thinned to the original cell number/ml and incubated an additional 3 days without the further addition of LTD4. Cytospin slides were stained for morphological analysis using May-Grünwald/Giemsa stain. Cells were monitored for superoxide production by following NBT reduction using TPA/NBT, as described in the methods section, and counterstaining with Safranin. Reprinted from Hazel et al. (24) with permission of AlphaMed Press.
Figure 7. Effects of LTD4 and HQ on proliferation of 32D cells in the presence or absence of IL-3. Cells (2.5X105/ml) were pretreated with 2 
Figure 8. Profile of stage-specific differentiation induced by LTD4 and HQ treatment of 32D cells in the absence of IL-3. Cells (2.5X105/ml) were treated with LTD4 or HQ in the absence of IL-3, as described for Figure 7. Samples of each culture were collected every second day and Cytospin preparations made and stained using May-Grünwald/Giemsa as described in the methods section. Morphologic analysis and determination of the percentages for stage-specific differentiation were performed by counting 200 cells on each of triplicate slides per sample and determining an average percentage of cells at each stage of differentiation. Percentage of total differentiation was calculated by totaling the percentages of stage-specific differentiation for each sample. P, promyelocytes; My, myelocytes; M, metamyelocytes; and Ma, mature segmented granulocytes.
