dicumarol and 1-2-dithiol-3-thione

Oral exposure of DBA/2 mice to benzo[a]pyrene (BP) has been shown to result in hematotoxicity which is manifested as aplastic anemia and leukemia. Since normal hematopoiesis is regulated by bone marrow stromal cells, in this study we have characterized the bone marrow stromal toxicity induced by BP and BP-derived metabolites, particularly quinones. Incubation of stromal cells with various concentrations of BP-1,6-, 3,6-, 6,12-, or 7,8-quinone for 24 hr resulted in a significant decrease of cell survival in a concentration-dependent manner, while cells treated with BP or BP-7,8-dihydrodiol did not exhibit any significant loss of cell survival. Among the BP quinones examined, BP-1,6-quinone was the most cytotoxic to stromal cells. The cytotoxicity induced by BP-1,6-quinone also exhibited a time-dependent relationship. Pretreatment of stromal cells with 1,2-dithiole-3-thione (D3T) resulted in a significant induction of both cellular reduced glutathione (GSH) content and quinone reductase (QR) activity in a concentration-dependent manner. However, D3T pretreatment did not offer any protection against BP-1,6-quinone-induced toxicity. Furthermore, dicumarol, a potent inhibitor of QR, or buthionine sulfoximine, a specific inhibitor of GSH biosynthesis, did not potentiate BP-1,6-quinone-induced cytotoxicity was not altered. However, incubation of stromal cells with BP-1,6-quinone resulted in a significant depletion of cellular ATP content and mitochondrial morphological changes, which preceded the loss of cell survival. In addition to BP-1,6-quinone, other cytotoxic BP quinones also exhibited a capacity to deplete cellular ATP level in stromal cells, while BP, which was not cytotoxic to stromal cells, did not elicit any significant decrease in cellular ATP level. These observations suggest that mitochondria may be a potential target of BP quinones. Overall, the above results indicate that neither cellular GSH and QR nor reactive oxygen species appear to be involved in BP quinone-induced stromal cell injury and that BP quinones may elicit cytotoxicity to stromal cells through directly disrupting mitochondrial energy metabolism.

Topics: Adenosine Triphosphate; Anemia, Aplastic; Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Benzo(a)pyrene; Benzopyrenes; Bone Marrow; Bone Marrow Cells; Buthionine Sulfoximine; Cell Survival; Cells, Cultured; Dicumarol; Disease Models, Animal; Dose-Response Relationship, Drug; Glutathione; Leukemia, Experimental; Male; Methionine Sulfoximine; Mice; Mice, Inbred DBA; Mitochondria; NAD(P)H Dehydrogenase (Quinone); Quinones; Stromal Cells; Superoxide Dismutase; Thiones; Thiophenes

In this study, we have characterized quinone reductase (QR), glutathione (GSH), glutathione S-transferase (GST) and their induction by a chemoprotector, 1,2-dithiole-3-thione (D3T), in the human myeloid cell lines ML-1 and HL-60. In addition, we also examined the toxicity of hydroquinone (HQ), a benzene metabolite, to these two cell lines. Both of the cell lines contain a basal level of cellular GSH, which is similar in the two cell lines. Although ML-1 cells contain much higher QR specific activity than HL-60 cells, which are relatively QR deficient, the GST specific activity of ML-1 cells is 1.8 times less than that of HL-60 cells. Immunoblot experiments showed that the GST in these two cell lines is GST pi. In addition, HL-60 cells exhibit 4.5 times more myeloperoxidase specific activity than ML-1 cells. Inclusion of D3T in the cultures could induce significant increases in cellular GSH content and QR activity, but not GST activity in either cell line. Treatment with HQ caused both inhibition of cell proliferation and loss of cell viability in these two myeloid cell lines. HQ treatment also resulted in a significant depletion of cellular GSH, which preceded the loss of cell viability. Pretreatment of both cell lines with buthionine sulfoximine, an inhibitor of GSH biosynthesis, markedly increased HQ-induced toxicity. In contrast, the presence of dicumarol, a QR inhibitor, failed to potentiate HQ-induced toxicity in ML-1 cells. On the other hand, pretreatment of these two myeloid cell lines with D3T significantly protected against HQ-induced inhibition of cell proliferation and cell death. Therefore, the above results suggest that GSH but not QR is an important factor involved in the toxicodynamics of HQ in these myeloid cells.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Agents; Buthionine Sulfoximine; Dicumarol; Drug Synergism; Enzyme Induction; Glutathione; Glutathione Transferase; Humans; Hydroquinones; Leukemia, Myeloid; Methionine Sulfoximine; Models, Chemical; NAD(P)H Dehydrogenase (Quinone); Peroxidase; Thiones; Thiophenes; Tumor Cells, Cultured

Stromal cells from bone marrow are susceptible to toxicity induced by several redox-active metabolites of benzene, including hydroquinone (HQ). We have previously shown that tert-butyl-hydroquinone (tBHQ) can induce quinone reductase (QR) in bone marrow stroma as well as protect stromal cells against HQ-induced toxicity. Current studies investigate the underlining mechanisms of chemoprotection against HQ in DBA/2- and C57Bl/6-derived bone marrow stromal cells. The chemoprotector 1,2-dithiole-3-thione (DTT) has been used in these studies due to tBHQ toxicity to stromal cells at higher concentrations. Pretreatment of cells with DTT prior to HQ administration protected cells against HQ-induced toxicity. DTT induced QR activity in a dose-dependent manner in stromal cells from both strains of mice. However, there were no corresponding changes in glutathione transferase activity. DTT also increased cytosolic glutathione (GSH) concentrations by approximately 85% in both strains. Since bone marrow stroma consists primarily of fibroblasts and macrophages, we also evaluated QR activity in the separate cell types from the two strains of mice. There were differences in basal and DTT-induced QR activity between fibroblasts and macrophage cells derived from the same strain of mice, as well as the expected differences between strains. Additionally, dicoumarol, an inhibitor of QR activity, potentiated HQ-induced toxicity in both strains of bone marrow stromal cells. Thus, cellular glutathione, QR activity, and their inducibility by chemoprotective agents such as DTT may prove to be important factors in chemically induced bone marrow toxicity and carcinogenicity.

Topics: Animals; Antineoplastic Agents; Bone Marrow; Bone Marrow Cells; Dicumarol; Drug Synergism; Enzyme Induction; Fibroblasts; Glutathione; Glutathione Transferase; Hydrogen Peroxide; Hydroquinones; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Peroxidases; Quinone Reductases; Receptors, Aryl Hydrocarbon; Receptors, Drug; Thiones; Thiophenes; Time Factors