Biotransformation of para-aminobenzoic acid and salicylic acid by PMN.

Para-aminobenzoic acid (PABA) is an essential cofactor for the production of folic acid in bacteria and has mild anti-inflammatory activity. We have recently reported that salicylic acid and benzoic acid are oxidized by stimulated granulocytes Polymorphonuclear Neutrophils (PMN). The oxidation of salicylate appears mediated by a potent oxygen metabolite generated during the respiratory burst which is dependent primarily on superoxide (O2-) for its production. These background studies with the salicylate group of drugs suggested that PABA might be similarly metabolized by PMN. In these studies, we demonstrate that PABA is metabolized by stimulated PMN. However, in contrast to the biochemical mechanism involved in the metabolism of salicylate, our scavenger studies indicate that PABA is metabolized primarily by the myeloperoxidase pathway. Our results may explain the mild anti-inflammatory actions of the drug and suggest that the degradation of PABA by PMN at an inflammatory site may limit the availability of PABA for bacterial growth.

Glucose metabolism of hairy cells.

Hairy cell leukemia is a malignant B-cell disorder characterized by splenomegaly and pancytopenia. The malignant cell is morphologically unique and characterized by fine cytoplasmic projections. Although studies of the cell have revealed important information about its proliferative capacity, cell surface, and membrane composition, less is known about the metabolic characteristics of the cell. We have previously investigated the oxidative metabolism of the hairy cell and have suggested that hairy cells might have a unique glucose metabolism compared to normal lymphocytes. This is indicated by a high rate of [6-14C]glucose oxidation in short-term culture consistent with an active Kreb's cycle and a high ratio of [6-14C]glucose oxidation to [1-14C] glucose oxidation. In this study, we evaluated an additional group of patients with hairy cell leukemia prior to or after treatment with the experimental drug 2'-deoxycoformycin (dCF). We found that in seven of eight patients the leukemic cells had a pattern similar to that previously described and that all of these seven patients had a significant response to therapy. The cells of the eighth patient had minimal Kreb's cycle activity, and at the time of study the patient was resistant to therapy with dCF. The metabolic activity of hairy cells may distinguish them from other lymphoid populations and may be a marker for sensitivity to dCF.

Interleukin-2 therapy enhances salicylate oxidation by blood granulocytes.

These studies determined the effect of interleukin-2 (IL-2) immunotherapy on the oxidative metabolism of the blood granulocytes of eight patients with metastatic renal cancer. We quantitated the rate of the hexose monophosphate shunt activity (HMPS), hydrogen peroxide (H2O2) production, and salicylate oxidation of the unstimulated and phorbol myristate acetate (PMA)-stimulated granulocyte cultures before, during, and after a 5-day continuous infusion of IL-2. There was no change in the rate of HMPS activity. However, the rate of salicylate oxidation of the unstimulated and PMA-stimulated cultures of these patients was significantly increased after the therapy was complete. Overall, there was no increase in the rate of H2O2 production, although the PMA-stimulated cultures of three of eight patients had a twofold higher production of H2O2 after treatment compared with the pretreatment values. The enhanced rate of salicylate oxidation by the granulocytes after treatment indicates that these cells were "stimulated" in vivo to produce a potent oxidant, which is most likely hydroxyl radical or an oxidant of comparable activity. Further, the granulocytes were primed ("activated"), since they had an augmented response to PMA. IL-2 did not stimulate the oxidative metabolism of granulocyte cultures in vitro, suggesting that the IL-2 effect in vivo is not a direct one. Our results indicate that IL-2 immunotherapy is associated with the activation of blood granulocyte oxidative metabolism and that these activated granulocytes may be related to some of the toxic side effects of IL-2 therapy such as the capillary leak syndrome. Further oxidant injury to the granulocytes may explain the reported defect in chemotaxis.

Hydroxylation of salicylate by activated neutrophils.

Salicylates are metabolized in vivo to hydroxylated compounds, including 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid (gentisic acid). The present study hypothesized that activated neutrophils represent one pathway for salicylate hydroxylation. Human neutrophils were incubated in medium containing 10 mM salicylate and stimulated with phorbol myristate acetate (PMA) for 1 hr. The cell-free supernatant fractions were analyzed by HPLC. Neutrophils (1 x 10(6) cells) produced 55 +/- 11 ng of gentisic acid. Neutrophils also produced smaller quantities of 2,3-dihydroxybenzoic acid. Antioxidant inhibitor experiments indicated that superoxide dismutase (SOD), heme protein inhibitors, and glutathione blocked gentisic acid formation, whereas catalase, mannitol, and deferoxamine failed to inhibit. Experiments with the reagent hypochlorous acid (HOCl) and the model myeloperoxidase (MPO) enzyme system did not support a role for the MPO pathway in gentisic acid formation. These findings demonstrate that activated neutrophils can hydroxylate salicylate by an unknown pathway. This pathway may contribute to the increased recovery of hydroxylated salicylates in patients with inflammatory disorders.

Effect of dimethylthiourea on the neutrophil myeloperoxidase pathway.

The sulfur-centered compound dimethylthiourea (DMTU) affords antioxidant protection in animal models of acute lung injury, an effect that has been attributed to its OH. scavenging properties. Although DMTU can also react with H2O2 in certain experimental systems, the effect of DMTU on the neutrophil myeloperoxidase (MPO) pathway has not been studied. DMTU (1-10 mM) completely blocked stable oxidants and hypochlorous acid formation by phorbol myristate acetate- and zymosan-stimulated neutrophils. DMTU also provided complete inhibition when incubated with cell-free supernatants after the formation of the MPO products. DMTU prevented the oxidative inactivation of alpha 1-antitrypsin by neutrophil-stable oxidants. Evidence that DMTU was oxidized by the MPO products was obtained by titration of oxidized DMTU with reduced glutathione. Surprisingly, supernatants from cells incubated with DMTU (10 mM) consumed two- to threefold higher amounts of reduced glutathione than supernatants from cells incubated with taurine (15 mM). Metabolic studies with stimulated neutrophils and experiments with the MPO enzyme system in a cell-free system suggested that DMTU acts by scavenging the products of the MPO pathway rather than by blocking H2O2 production in the intact cell. These findings demonstrate that DMTU blocks the neutrophil MPO pathway in addition to its known ability to scavenge other reactive O2 species. The capacity of DMTU to scavenge MPO products may explain some of its protective effects in acute lung injury.

Activation and deactivation of guinea pig peritoneal eosinophils during chronic polymyxin B stimulation.

Eosinophils exhibit different levels of oxidative metabolism depending on their site of origin and various host factors that may influence metabolism. The present study examined the time course of eosinophil oxidative metabolism in animals undergoing chronic peritoneal stimulation. Eosinophils were purified from the peritoneal exudates of guinea pigs stimulated with weekly polymyxin B and saline peritoneal lavage. 14C-1- and 14C-6-glucose oxidation and H2O2 production were measured at week 0 and at various time points throughout 43 weeks of stimulation. Baseline oxidative metabolism of eosinophils was relatively high throughout the time course, but then declined sharply after 32 weeks. These "deactivated" cells that were recovered after 32 weeks also failed to respond to phorbol myristate acetate (PMA) or opsonized zymosan. Electron microscopy did not reveal significant differences between deactivated eosinophils and cells from earlier time points. These findings document the time course of eosinophil activation and deactivation in this model and suggest that metabolic heterogeneity of eosinophils can occur over time in response to a chronic stimulus.

Effect of O2 partial pressure on the myeloperoxidase pathway of neutrophils.

Neutrophils recruited to different tissues undergo respiratory burst activity at widely different PO2 levels. The present study investigated the in vitro effects of PO2 on neutrophil oxidative metabolism. When neutrophils were stimulated with either zymosan or phorbol myristate acetate (PMA) under different PO2's (0-700 Torr), hexose monophosphate shunt activity, H2O2, and hydroxyl radical (OH.) production were directly related to the level of PO2. Neutrophils functioned surprisingly well at PO2's as low as 10 Torr, where metabolic burst activity was prolonged and usually exceeded 50% of maximal values. The production of neutrophil stable oxidants and hypochlorous acid (HOCl) by zymosan-stimulated neutrophils was also directly related to PO2. In contrast, the production of stable oxidants and HOCl by PMA-stimulated neutrophils was significantly higher at 10 Torr compared with 700 Torr. The decrease in stable oxidant production by PMA-stimulated neutrophils at elevated PO2's was explained by both increased destruction of stable oxidant products and by decreased availability of the precursor HOCl. Superoxide dismutase and the OH. scavenger benzoate partially prevented the fall in stable oxidants at elevated PO2's. Measurements of stable oxidants in PMA-stimulated supernates generated at 10 and 700 Torr correlated with the ability of these supernates to decrease the elastase inhibitory capacity of the serum antiprotease alpha 1-antitrypsin. These findings suggest that different PO2's alter the magnitude and pattern of neutrophil oxidative metabolism.

Oxidation of salicylates by stimulated granulocytes: evidence that these drugs act as free radical scavengers in biological systems.

Although salicylates have been used for centuries as treatment of inflammatory diseases, the mechanism of action of these drugs is still not clear. Aspirin (acetylsalicylic acid) and other nonsteroidal anti-inflammatory drugs (NSAID) inhibit prostaglandin biosynthesis, a property that appears to explain part of their anti-inflammatory activity. However, this mechanism does not appear to explain the anti-inflammatory properties of salicylic acid, which is a major metabolite of ASA in vivo. Results of prior studies in our laboratory have established that benzoic acid, the parent compound of the salicylate group of drugs, is decarboxylated and hydroxylated by the hydroxyl free radical (OH.) produced by stimulated granulocytes. These observations suggested that salicylates might be similarly metabolized by granulocytes. If so, the capacity of salicylates to rapidly react with OH. might relate directly to their known anti-inflammatory properties. Preliminary experiments established that salicylic acid and aspirin were decarboxylated by the hydroxyl free radical generated by the enzyme system xanthine-xanthine oxidase. We then studied the metabolism of salicylates by human granulocytes. Unstimulated granulocyte suspensions did not oxidize ASA or salicylic acid. However, suspensions stimulated by opsonized zymosan particles rapidly oxidized both substrates in pharmacological concentrations. The rate of oxidation of salicylic acid was 16-fold higher than benzoic acid, whereas the rate of oxidation of ASA was four-fold higher. The reaction was oxygen dependent and could be inhibited by known hydroxyl scavengers, particularly dimethylthiourea. The reaction could also be inhibited by superoxide dismutase and azide, indicating that O-2 and heme or an iron-dependent enzyme were required for the reaction. The reaction was not impaired by compounds known to react with the HOCL and the chloramines generated by stimulated PMN. Furthermore, salicylic acid in high concentrations did not impair the HMPS pathway, the production of O-2 or the production of H2O2 by granulocytes. These data provide evidence that salicylates are rapidly oxidized by the hydroxyl free radical produced by granulocytes and not O-2, H2O2, or HOCL. This capacity of salicylates to react rapidly and selectively react with OH. may directly relate to their anti-inflammatory properties. In addition, results of our experiments indicate that stimulated granulocytes acquire the capacity to metabolize these drugs. Therefore, several metabolites of salicylates may be produced at a site of inflammation, all of which may have altered biological activity compared with the parent compound.

Mechanisms for the oxidation of reduced gluthathione by stimulated granulocytes.

We have reported previously that human granulocytes have an irreversible fall in their endogenous reduced soluble sulfhydryls following zymosan stimulation. In the present study, we demonstrate that stimulated granulocytes release one or more reactive oxygen species (ROS) with the capacity to oxidize reduced glutathione (GSH). One or more of these compounds is stable enough to be detected in the supernatant. The formation of these stable oxidants appears to require H2O2 and heme or a heme-containing enzyme. However, once formed, the compound reacts with GSH without these factors. The ROS is not superoxide or hydroxyl radical, since neither superoxide dismutase nor the hydroxyl scavengers, mannitol and benzoic acid, change the rate of the reaction. Methionine has recently been demonstrated to be oxidized to a sulfoxide by a reactive oxygen species that is dependent on H2O2 and heme for its production. We found that methionine could directly react with the same ROS that degrades GSH. The ROS also has the capacity to oxidize iodide and fix halogen to proteins. Our data indicate that stimulated granulocytes release a ROS with the capacity to oxidize GSH, react with methionine, and oxidize and fix I- to protein. The compound, therefore, appears dependent on H2O2 and the myeloperoxidase system for its production, and is either hypochlorous acid (HOCI) or a compound derived from HOCI, such as a chloramine. The capacity of GSH to react with this ROS suggests an additional role for this tripeptide in cellular protection against oxidant injury.

Effect of 2,3-dihydro-1H-imidazo [1,2-b]pyrazole (IMPY) on the metabolism of human red cells.

2,3 dihydro-IH-imidazo (1,2-b) pyrazole (IMPY) is a potential chemotherapeutic agent known to inhibit cellular DNA synthesis by blocking ribonucleotide reductase. During Phase I clinical studies with IMPY, patients developed a dose dependent hemolytic anemia possibly secondary to oxidant damage to the RBC. We therefore studied the effect of IMPY on the metabolism of human RBC's in vitro. IMPY, in clinically achievable concentrations, stimulated the hexose monophosphate shunt (HMPS) pathway of RBC's. This was associated with the generation of reactive oxygen species as demonstrated by the glutathione (GSH) instability and enhanced formate oxidation of RBC's incubated with the drug. In addition, the GSH concentration of the red cells of a patient fell during a continuous infusion of IMPY. These effects of IMPY on red cell metabolism in vitro and in vivo are similar to those of drugs known to cause oxidative damage to this cell. The capacity of IMPY to act as an oxidant could explain the hemolytic anemia seen in patients receiving this drug.