calpain and hydroquinone

Chronic exposure of humans to benzene causes severe bone marrow cell depression leading to aplastic anemia. Marrow stromal macrophage dysfunction and deficient interleukin-1 production has been reported for patients with severe aplastic anemia. The stromal macrophage, a target of benzene toxicity, is involved in hematopoietic regulation through the synthesis of several cytokines including interleukin-1, which is required for production by stromal fibroblasts of a number of cytokines required for the survival of hematopoietic progenitor cells. We have previously demonstrated that hydroquinone, a major toxic metabolite of benzene in marrow, prevents the proteolytic conversion of 31 kDa pre-interleukin-1 alpha to the 17 kDa cytokine by calpain in purified murine stromal macrophages. Furthermore, stromal macrophages from benzene-treated mice produce the 31 kDa pre-interleukin-1 alpha when stimulated in culture with endotoxin, but cannot convert the precursor to interleukin-1 alpha. In this report, we show that 1,4-benzoquinone, the oxidation product of hydroquinone in the cell, causes a concentration-dependent inhibition of highly purified human platelet calpain with an IC50 of 3 microM. Hydroquinone also inhibits the processing of pre-interleukin-1 beta by interleukin-1 beta convertase. The addition of 2 microM hydroquinone to B1 cells that undergo autocrine stimulation by interleukin-1 beta resulted in the cessation of autocrine cell growth and interleukin-1 beta secretion into the culture medium, as determined by Western immunoblots of the culture supernatants. Purified converting enzyme treated with 3 microM benzoquinone was incapable of converting 31 kDa recombinant pre-interleukin-1 beta to the 17 kDa mature cytokine as analyzed by polyacrylamide gel electrophoresis and Western immunoblotting. These findings support our observations in a mouse model that benzene-induced bone marrow cell depression results from a lack of interleukin-1 alpha subsequent to an inhibition by benzoquinone of calpain, the protease required for converting pre-interleukin-1 alpha to active cytokine. The results may provide a basis for studying benzene-induced aplastic anemia in a mouse model.

Topics: Benzene; Benzoquinones; Blotting, Western; Calpain; Caspase 1; Cell Division; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Humans; Hydroquinones; Indomethacin; Interleukin-1; Macrophages; Protein Precursors; Recombinant Proteins; Tumor Cells, Cultured

Benzene is an important industrial chemical known to produce hematotoxicity in mice and humans. Hydroquinone, a major metabolite of benzene, inhibits conversion of the precursor form of IL1 alpha (pre-IL1 alpha) to IL1 alpha in murine bone marrow-derived macrophages in vitro, and a similar effect can be demonstrated in vivo after treatment of mice with benzene. The protease which converts pre-IL1 alpha to IL1 alpha is calpain. We examined decreases in calpain content in bone marrow-derived macrophages as a possible mechanism underlying hydroquinone-induced decreases in pre-IL1 alpha conversion. Hydroquinone, at concentrations which were not overtly cytotoxic, decreased total calpain activity in macrophages by 10-30%. Using immunoblot analysis macrophage calpain II levels were shown to be decreased by approximately 50% after treatment with hydroquinone. Under the same conditions, no changes were observed in calpain I content using immunoblot analysis. These data show that decreased calpain II content represents a potential mechanism of hydroquinone-induced inhibition of pre-IL1 alpha processing, and may contribute to benzene-induced alterations in bone marrow stromal cell function and myelotoxicity.

Topics: Animals; Bone Marrow Cells; Calpain; Hydroquinones; Interleukin-1; Macrophages; Male; Mice