Induction of granulocytic differentiation in a mouse model by benzene and hydroquinone.

Chronic exposure of humans to benzene causes acute myelogenous leukemia (AML). The studies presented here were undertaken to determine whether benzene, or its reactive metabolite, hydroquinone (HQ), affects differentiation of myeloblasts. Benzene or HQ administered to C57BL/6J mice specifically induced granulocytic differentiation of myeloblasts. The ability of these compounds to induce differentiation of the myeloblasts was tested directly using the murine interleukin 3 (IL-3)-dependent 32D.3 (G) myeloblastic cell line, and the human HL-60 promyelocytic leukemia cell line. We have previously shown that benzene treatment of HL-60 myeloblasts activates protein kinase C (PKC) and upregulates the 5-lipoxygenase (LPO) pathway for the production of leukotriene D4 (LTD4), an essential effector or granulocytic differentiation. Differentiation was prevented by sphinganine, a PKC inhibitor, and, as shown here, by LPO inhibitors and LTD4 receptor antagonists. Benzene or HQ also induces differentiation in 32D.3 (G) myeloblasts. Both compounds interact with cellular signaling pathways normally activated by granulocyte colony stimulating factor (G-CSF) and can replace the requirement for G-CSF. While IL-3 induces a growth response in 32D.3 (G) cells, G-CSF has been shown to provide both growth and differentiated signals. Both HQ and LTD4 induce differentiation and synergize with IL-3 for growth; however, neither supports growth in the absence of IL-3. Benzene, like HQ, also provides a differentiation signal for 32D cells; however, it has no effect on their growth. Unlike G-CSF, benzene, or LTD4, each of which stimulates terminal differentiation; HQ blocks differentiation at the myelocyte stage, allowing only a small percentage of progenitors to proceed to mature segmented granulocytes. Benzene- and G-CSF-induced differentiation were prevented by the additional of either LPO inhibitors or LTD4 receptor antagonists, indicating that benzene, like G-CSF, upregulates LTD4 production. Hydroquinone-induced differentiation was not affected by the LPO inhibitors, but only by the specific receptor antagonists. Thus HQ appears to obviate the requirement for LTD4 by activating the LTD4 receptor directly.

Hydroquinone, a bioreactive metabolite of benzene, inhibits apoptosis in myeloblasts.

Hydroquinone (a major marrow metabolite of the leukemogen, benzene) induces incomplete granulocytic differentiation of mouse myeloblasts to the myelocyte stage, and also causes an increase in the number of myelocytes. This was confirmed using the normal interleukin 3 (IL-3)-dependent mouse myeloblastic 32D cell line. The hydroquinone-induced twofold increase in the number of IL-3-treated myelocytes does not result from stimulation of IL-3-induced proliferation. Hydroquinone's ability to effect this increase through an inhibition of apoptosis was investigated using mouse 32D and human HL-60 myeloblasts. Apoptosis induced by staurosporine treatment (0.5-1.0 microM) of HL-60 cells (50%) and 32D cells (15%) or by IL-3 withdrawal from 32D myeloblasts was determined by monitoring the development of characteristic morphological features and confirmed by the appearance of a typical nucleosomal DNA ladder upon agarose gel electrophoresis. Concentrations of hydroquinone (1-6 microM) that induce differentiation in 32D myeloblasts caused a concentration-dependent inhibition of staurosporine-induced apoptosis in both cell lines, with a 50% inhibitory concentration of 3 microM, and prevented apoptosis in IL-3-deprived 32D cells. Hydroquinone inhibition of apoptosis in myeloblasts, like hydroquinone-induced granulocytic differentiation, required myeloperoxidase-mediated oxidation of hydroquinone to its reactive species, p-benzoquinone, and was inhibited 50% by the peroxidase inhibitor, indomethacin (20 microM). p-benzoquinone (3 microM) was shown to cause a 50% inhibition of CPP32, an IL-1 beta-converting enzyme/Ced-3 cysteine protease involved in the implementation of apoptosis and present in myeloid cells. The ability of hydroquinone to induce a program of differentiation in the myeloblast that proceeds only to the myelocyte stage coupled with its ability to inhibit the CPP32 protease and, thereby, apoptosis of the proliferating myelocytes, may have important implications for benzene-induced acute myeloid leukemia.

Induction of granulocytic differentiation in myeloblasts by hydroquinone, a metabolite of benzene, involves the leukotriene D4 receptor.

Chronic exposure of humans to benzene (BZ), a Class I carcinogen, causes acute myelogenous leukemia, possibly via its bone marrow metabolite, hydroquinone (HQ). The ability to alter cytokine-dependent growth and differentiation in hematopoietic stem or progenitor cells appears to be a property of agents with leukemogenic potential. We have previously reported that BZ and HQ specifically stimulate granulopoiesis in mice and cause granulocytic differentiation in normal murine interleukin (IL)-3-dependent, granulocyte colony-stimulating factor (G-CSF)-inducible 32D myeloblasts. BZ induces granulocytic differentiation by upregulating the production of leukotriene D4 (LTD4), an essential intracellular mediator of G-CSF signaling. We report here that HQ (0.5-4.0 microM), as well as LTD4 (1 nM-10 microM), causes a concentration-dependent induction of granulocytic differentiation in 32D myeloblasts. Unlike LTD4, which induces terminal granulocytic differentiation, HQ undergoes a myeloperoxidase-dependent oxidation to bioreactive p-benzoquinone (BQ), which induces differentiation predominantly to the myelocyte stage. Studies with the highly specific LTD4 receptor antagonist, MK-571, suggest that BQ induces granulocytic differentiation in myeloblasts by activating the LTD4 receptor, thus obviating the requirement for LTD4. This was confirmed by the demonstration that HQ, in the presence of LTD4, shifts the stage-specific pattern of terminal differentiation induced by LTD4 to the incomplete (myelocyte) profile induced by HQ. The inability of HQ to induce a complete program of terminal granulocytic differentiation in myeloblasts, as well as its ability to compete with induction by LTD4, may have a bearing on the leukemogenic potential of BZ.

Induction of granulocytic differentiation in myeloblasts by 17-beta-estradiol involves the leukotriene D4 receptor.

17-beta-Estradiol (beta E) causes granulocytic differentiation and neutrophilia in mice. However, the presence of estrogen receptors in myeloblasts and granulocytic progenitor cells has not been reported. beta E can be converted to a bioreactive species, estradiolquinone. We have previously shown that hydroquinone (HQ), via conversion to bioreactive p-benzoquinone (BQ), causes neutrophilia in mice and induces granulocytic differentiation in myeloblasts through interaction with the leukotriene D4 (LTD4) receptor. Therefore, we tested whether beta E could be oxidized by a myeloperoxidase-mediated reaction to a bioreactive intermediate, which might, in turn, induce granulocytic differentiation in mouse myeloblasts by activating the LTD4 receptor, thus obviating the need for LTD4, the downstream intracellular mediator of granulocyte colony-stimulating factor (G-CSF)-induced signal transduction. The interleukin (IL)-3-dependent, G-CSF-inducible normal mouse myeloblastic cell line, 32D cl 3(G), was used to determine the ability of beta E to induce terminal granulocytic differentiation in myeloblasts. Morphological analysis of stage-specific granulocytic differentiation indicated that beta E was capable of the concentration- (10(-8)-10(-4)M) and time-(6d) dependent induction of a complete program of terminal granulocytic differentiation in myeloblasts similar to that seen with G-CSF or LTD4. beta E-induced granulocytic differentiation was prevented by the peroxidase inhibitor, indomethacin, and was completely and competitively inhibited in the presence of a specific LTD4 receptor antagonist, MK-571, suggesting that a bioreactive form of estradiol, such as estradiolquinone, is interacting with the receptor. beta E was shown to cause a similar concentration-dependent induction of granulocytic differentiation in human HL-60 myeloblasts that was also inhibited by the receptor antagonist. Biological effects of beta E in nontarget tissues may result from the interaction of bioreactive estradiolquinone with critical cellular macromolecules involved in normal cellular signaling pathways.

Benzene and its metabolite, hydroquinone, induce granulocytic differentiation in myeloblasts by interacting with cellular signaling pathways activated by granulocyte colony-stimulating factor.

Chronic exposure of humans to benzene (BZ) causes acute myelogenous leukemia. These studies determined whether BZ, or its reactive metabolite, hydroquinone (HQ), affect differentiation of myeloblasts. BZ or HQ administered to C57BL/6J mice specifically induced terminal granulocytic differentiation of myeloblasts. The ability of the compounds to induce differentiation of the myeloblast was tested directly using the murine interleukin 3 (IL-3)-dependent myeloblastic cell line, 32D.3 (G) and the human HL-60 promyelocytic leukemic cell line. Treatment of HL-60 myeloblasts with BZ activated protein kinase C and upregulated the 5-lipoxygenase (LPO) pathway for the production of leukotriene D4 (LTD4), an essential effector of granulocytic differentiation. Differentiation was prevented by sphinganine, a kinase C inhibitor, as well as by LPO inhibitors and LTD4 receptor antagonists. BZ and HQ also induced differentiation in 32D.3 (G) myeloblasts. Both compounds interact with cellular signaling pathways activated by granulocyte colony-stimulating factor (G-CSF) and thus replace the requirement for G-CSF. IL-3 induces a growth response, whereas G-CSF provides both growth and differentiation signals. BZ does not induce growth in the absence of IL-3, but provides a differentiation signal. Both HQ and LTD4 induce differentiation and synergize with IL-3 for growth, however, neither support growth in the absence of IL-3. BZ-induced 32D cells showed a gradual progression of progenitor differentiation to granulocytes similar to that seen with G-CSF or LTD4. HQ blocks differentiation at the myelocyte stage; only a small percentage of progenitors proceed to granulocytes. BZ, like G-CSF, upregulates LTD4 production, whereas HQ obviates the requirement for LTD4 by activating the LTD4 receptor.