Hydroquinone, a metabolite that accumulates in bone marrow following benzene exposure, was oxidized by prostaglandin H synthase (PHS) to 1,4-benzoquinone, which was measured by HPLC with reductive electrochemistry. Hydroquinone metabolism in the presence of cysteine generated a thiol adduct, which was identified as the monosubstituted cysteine conjugate of hydroquinone by HPLC with oxidative electrochemical and radiochemical detection. The time-dependent formation of both 1,4-benzoquinone and the monocysteine-hydroquinone conjugate was monitored spectrophotometrically at 250 and 305 nm, respectively. Monocysteine-hydroquinone was formed at rates similar to 1,4-benzoquinone formation in reactions without cysteine, suggesting that 1,4-benzoquinone or its semiquinone intermediate is rapidly binding to sulfhydryls. The PHS-catalyzed activation of hydroquinone to 1,4-benzoquinone or its thiol conjugate required the presence of either arachidonic acid or H2O2. The oxidative metabolism of hydroquinone also resulted in the formation of a reactive product(s) that irreversibly bound to DNA. This binding was time dependent and did not occur in the presence of heat-inactivated PHS. Metabolite(s) generated during hydroquinone oxidation also induced single-strand breaks in Bluescript plasmid DNA. The PHS/arachidonic acid catalyzed metabolism of hydroquinone to 1,4-benzoquinone and to product(s) that bound to sulfhydryls and DNA and caused strand breaks in DNA was prevented by indomethacin, an inhibitor of PHS cyclooxygenase. Because prostaglandin synthesis is elevated in bone marrow following benzene exposure and inhibitors of PHS cyclooxygenase prevent benzene-induced myelotoxicity, the activation of hydroquinone by PHS represents a possible mechanism for benzene's effects.

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In previous experiments we have shown that acute (30 minutes) exposure to phorbol esters or other protein kinase C activators causes increased transepithelial permeability, specifically by the increased paracellular permeability through tight junctions. However, the role of protein kinase C activators in carcinogenesis is predicted upon a chronic exposure of an effective dose at frequent intervals for a prolonged period of time. We therefore sought to determine the effect of chronic phorbol ester exposure on transepithelial permeability by exposing cells of the polar renal epithelial cell line, LLC-PK1, to phorbol esters for time periods as long as 16 weeks. The following changes ensued: (1) after the initial drop in transepithelial resistance due to phorbol ester exposure, i.e., an increase in transepithelial permeability (in the acute phase of exposure), an adaptive response occurs as transepithelial resistances in chronically exposed cultures recover to approximately 50% of control values, (2) the cell sheets in chronically exposed cultures lose their acute responsiveness of transepithelial permeability to phorbol ester exposure, (3) cell sheet architecture changes as cells occasionally multilayer and actual polyp-like cell masses appear at high frequency, and (4) cytosolic protein kinase C activity decreases to 50% of control level with acute exposure and then is further decreased to less than 1% of control level in chronically treated cells; membrane-associated PKC activity is not as sharply decreased. The possible role of transepithelial permeability in carcinogenesis and the value of chronically treated epithelial cell cultures as a model for two-stage carcinogenesis are discussed.

Macrophages, an important cell-type of the bone marrow stroma, are possible targets of benzene toxicity because they contain relatively large amounts of prostaglandin H synthase (PHS), which is capable of metabolizing phenolic compounds to reactive species. PHS also catalyzes the production of prostaglandins, negative regulators of myelopoiesis. Studies indicate that the phenolic metabolites7of benzene are oxidized in bone marrow to reactive products via peroxidases. With respect to macrophages, PHS peroxidase is implicated, as in vivo benzene-induced myelotoxicity is prevented by low doses of nonsteroidal anti-inflammatory agents, drugs that inhibit PHS. Incubations of either '4C-phenol or '4C-hydroquinone with a lysate of macrophages collected from mouse peritoneum (> 95% macrophages), resulted in an irreversible binding to protein that was dependent upon H202, incubation time, and concentration of radiolabel. Production of protein-bound metabolites from phenol or hydroquinone was inhibited by the peroxidase inhibitor aminotriazole. Protein binding from '4C-phenol also was inhibited by 8 pM hydroquinone, whereas binding from '4C-hydroquinone was stimulated by 5 mM phenol. The nucleophile cysteine inhibited protein binding of both phenol and hydroquinone and increased the formation of radiolabeled water-soluble metabolites. Similar to the macrophage lysate, purified PHS also catalyzed the conversion of phenol to metabolites that bound to protein and DNA; this activation was both H202-and arachiodonic acid-dependent. These results indicate a role for macrophage peroxidase, possibly PHS peroxidase, in the conversion of phenol and hydroquinone to reactive metabolites and suggest that the macrophage should be considered when assessing the hematopoietic toxicity of benzene.