Drug residue formation from ronidazole, a 5-nitroimidazole. I. Characterization of in vitro protein alkylation

The metabolic activation of [14C]ronidazole by rat liver enzymes to metabolite(s) bound to macromolecules was investigated. The alkylation of protein by [14C]ronidazole metabolite(s) was catalyzed most efficiently by rat liver microsomes, in the absence of oxygen utilizing NADPH as a source of reducing equivalents. Based on a comparison of total ronidazole metabolized versus the amount bound to microsomal protein, approximately one molecule alkylates microsomal protein for every 20 molecules of ronidazole metabolized. Protein alkylation was strongly inhibited by sulfhydryl-containing compounds such as cysteine and glutathione whereas methionine had no effect. Based on HPLC analysis of ronidazole, cysteine was found not to inhibit microsomal metabolism of ronidazole ruling out a decrease in the rate of production of the reactive metabolite(s) as the mechanism of cysteine inhibition.     

Multi-step metabolic activation of benzene. Effect of superoxide dismutase on covalent binding to microsomal macromolecules,and identification of glutathione conjugates using high pressure liquid chromatography and field desorption mass spectrometry

Incubation of [¹⁴C]benzene or [¹⁴C]phenol with liver microsomes from untreated rats, in the presence of a NADPH-generating system, gave rise to irreversible binding of metabolites to microsomal macromolecules. For both substrates this binding was inhibited by more than 50% by addition of superoxide dismutase to the incubation mixtures. The decrease in binding was compensated for by accumulation of [¹⁴C]hydroquinone, indicating superoxide-mediated oxidation of hydroquinone as one step in the activation of benzene to metabolites binding to microsomal macromolecules. Since our previous work had shown that binding occurred mainly with protein rather than ribonucleic acid and was virtually completely prevented by glutathione, suggesting identity of metabolite(s) responsible for binding to protein and glutathione, a conjugate was chemically prepared from p-benzoquinone and reduced glutathione (GSH) and identified by field desorption mass spectrometry (FDMS) as 2-(S-glutathionyl) hydroquinone. Microsomal incubations, containing an NADPH-generating system, with benzene, phenol, hydroquinone or p-benzoquinone in the presence of [³H]glutathione or, alternatively, with [¹⁴C]benzene or [¹⁴C]phenol in the presence of unlabeled glutathione, were performed. All of these incubations gave rise to a peak of radioactivity eluting from the high pressure liquid chromatograph (HPLC) at a retention time identical to that of the chemically prepared 2-(S-glutathionyl) hydroquinone, whilst microsomal incubation of catechol in the presence of [³H]glutathione led to a conjugate with a very different retention time which was not observed after incubation of benzene or phenol. The microsomal metabolites of p-benzoquinone, hydroquinone and phenol thus eluting from the HPLC were further identified as the 2-(S-glutathionyl) hydroquinone by field desorption mass spectrometry. The glutathione adduct formed from benzene during microsomal activation eluted from HPLC with the same retention time and its mass spectrum also contained the molecular ion (MH⁺) (m/e 416) of this conjugate as an intense peak, but the fragmentation patterns did not allow definite assignments probably due to the considerably smaller amounts of ultimate reactive metabolites formed from this pre-precursor and thus relatively larger amounts of impurities.The results indicate that rat liver microsomes activate benzene via phenol and hydroquinone to p-benzosemiquinone and/or p-benzoquinone as quantitatively important reactive metabolites.     

Hydroquinone, a reactive metabolite of benzene, reduces macrophage-mediated immune responses

Hydroquinone is a toxic compound and a major benzene metabolite. We report that it strongly inhibits the activation of macrophages and associated cells. Thus, it suppressed the production of proinflammatory cytokines [tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-3, IL-6, IL-10, IL-12p40, IL-23], secretion of toxic molecules [nitric oxide (NO) and reactive oxygen species (ROS)] and the activation and expression of CD29 as judged by cell-cell adhesion and surface staining experiments. The inhibition was due to the induction of heme oxygenase (HO)-1 in LPS-activated macrophages, since blocking HO-1 activity with ZnPP, an HO-1 specific inhibitor, abolished hydroquinone's NO inhibitory activity. In addition, hydroquinone and inhibitors (wortmannin and LY294002) of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway had very similar inhibitory effects on LPS-induced and CD29-mediated macrophage responses, including the phosphorylation of Akt. Therefore, our data suggest that hydroquinone inhibits macrophage-mediated immune responses by modulating intracellular signaling and protective mechanisms.     

Metabolism of benzene and phenol in macrophages in vitro and the inhibition of RNA synthesis by benzene metabolites

Benzene may affect hemopoiesis by damaging the bone marrow stroma that provides the microenvironment for hemopoiesis. A possible target of benzene toxicity in the stroma is the macrophage, which is a major source of protein factors required for the proliferation and differentiation of progenitor cells. As an initial approach towards understanding whether benzene inhibits hemopoietic factor production in bone marrow stroma, the metabolism of benzene and phenol has been studied and the effect of benzene and its metabolites on macrophage RNA synthesis has been examined. Benzene is not metabolized in macrophages but phenol, the major metabolite of benzene in bone marrow, is converted by peroxidase in the macrophage to both free metabolites and species which covalently bind to cellular macromolecules. Benzene and its metabolites inhibited RNA synthesis in a dose-dependent manner, with 50% inhibitory concentrations of 5 × 10^–3M for benzene, 2.5 × 10^–3 M for phenol, 2.5 × 10^–5 M for hydroquinone, and 6 × 10^–6 M for p-benzoquinone; this inhibition was not attributable to loss of cell viability. Benzene, possibly by an inhibition of uridine transport into macrophages, and phenol, by its conversion to covalently binding species, inhibit RNA synthesis in macrophages and thus may inhibit the synthesis of colony stimulating factors required for hemopoiesis.Abbreviations CFU-G / M colony forming unit-granulocyte / macrophage - FCS fetal calf serum - IC₅₀ molar concentration causing 50% inhibition - PBS phosphate buffered saline     

Metabolism of hydroquinone by human myeloperoxidase: mechanisms of stimulation by other phenolic compounds.

Hydroquinone, a metabolite of benzene, is converted by human myeloperoxidase to 1,4-benzoquinone, a highly toxic species. This conversion is stimulated by phenol, another metabolite of benzene. Here we report that peroxidase-dependent hydroquinone metabolism is also stimulated by catechol, resorcinol, o-cresol, m-cresol, p-cresol, guaiacol, histidine, and imidazole. In order to gain insights into the mechanisms of this stimulation, we have compared the kinetics of human myeloperoxidase-dependent phenol, hydroquinone, and catechol metabolism. The specificity (Vmax/Km) of hydroquinone for myeloperoxidase was found to be 5-fold greater than that of catechol and 16-fold greater than that of phenol. These specificities for myeloperoxidase-dependent metabolism inversely correlated with the respective one-electron oxidation potentials of hydroquinone, catechol, and phenol and suggested that phenol- and catechol-induced stimulation of myeloperoxidase-dependent hydroquinone metabolism cannot simply be explained by interaction of hydroquinone with stimulant-derived radicals. Phenol (100 microM), catechol (20 microM), and imidazole (50 mM) did, however, all increase the specificity (Vmax/Km) of hydroquinone for myeloperoxidase, indicating that these three compounds may be stimulating hydroquinone metabolism by a common mechanism. Interestingly, the stimulation of peroxidase-dependent hydroquinone metabolism by other phenolic compounds was pH-dependent, with the stimulating effect being higher under alkaline conditions. These results therefore suggest that the interaction of phenolic compounds, presumably by hydrogen-bonding, with the activity limiting distal amino acid residue(s) or with the ferryl oxygen of peroxidase may be an important contributing factor in the enhanced myeloperoxidase-dependent metabolism of hydroquinone in the presence of other phenolic compounds.     

Detection and identification of sulfhydryl conjugates of p-benzoquinone in microsomal incubations of benzene and phenol

The glutathione and cysteine conjugates of p-benzoquinone are detected and conclusively identified in microsomal incubations of benzene and phenol using liquid chromatography/electrochemistry (LCEC). Identification of the compounds is based on retention time, electrochemical behavior and acid hydrolysis. The fact that both of these compounds can be detected easily in a benzene incubation provides further evidence that p-benzoquinone or the corresponding semiquinone is a product of benzene metabolism in vivo. The conjugation of p-benzoquinone with glutathione is predominantly a nonenzymatic process. This is illustrated by the fact that the addition of cytosolic glutathione-S-transferases do not significantly increase the amount of glutathione conjugate produced in a phenol incubation containing glutathione.The kinetic constants for phenol metabolism to hydroquinone by microsomal protein are calculated. As suspected, the rate of metabolism of phenol is significantly higher than the rate of benzene metabolism. The V_max for phenol metabolism was calculated to be 7.1 nmol/min/mg protein and the K_M was found to be 0.38 mM.The further oxidation of hydroquinone to p-benzoquinone appears to be primarily an enzymatic process. Incubations of just hydroquinone with glutathione at 37°C produced only a small amount of the glutathione conjugate. The addition of cytosolic protein increases the amount of p-benzoquinone produced about 10-fold. This could be due to the peroxidases found in that medium. The addition of microsomal protein and NADPH increases the amount of glutathione conjugate produced to over 100-fold of that produced nonenzymatically. This indicates that a microsomal enzyme is responsible for the oxidation of hydroquinone to p-benzoquinone in vitro and the subsequent covalent binding to macromolecules.     

1,4-Benzoquinone-based electrophoretic assay for glucose oxidase

The communication demonstrates feasibility of an enzyme microassay for glucose oxidase with 1,4-benzoquinone as an acceptor of electrons. The protocol uses the plug-plug mode of electrophoretically mediated microanalysis, with nanolitre injected volumes of enzyme and reactant solutions. The reactant and product, 1,4-benzoquinone and hydroquinone, are separated during the assay by differential binding to sulfated-beta-cyclodextrin used as additive to the phosphate buffer (pH 7) and monitored at selected wavelengths in their UV spectra. The assay covers glucose oxidase concentration from 0.01 to 0.1mgml(-1). Due to the strong UV absorbance of the both reactant and product, there is no need for use of a second enzyme (peroxidase) in the present assay.     

Metabolic activation of 1-naphthol and phenol by a simple superoxide-generating system and human leukocytes

Phenol and 1-naphthol, products of benzene and naphthalene biotransformation, are metabolized during O2- generation by xanthine oxidase/hypoxanthine and phorbol myristate acetate (PMA)-stimulated human neutrophils. The addition of 1-naphthol to xanthine oxidase/hypoxanthine incubations resulted in the formation of 1,4-naphthoquinone (1,4-NQ) whereas phenol addition yielded only small quantities of hydroquinone, catechol and a unidentified reducible product but not 1,4-benzoquinone. This formation of 1,4-NQ was dependent upon hypoxanthine, xanthine oxidase, and 1-naphthol and was inhibited by the addition of superoxide dismutase (SOD) demonstrating that the conversion was O2-mediated. During O2- generation by PMA-stimulated neutrophils, the addition of phenol interfered with luminol-dependent chemiluminescence and resulted in covalent binding of phenol to protein. Protein binding was 80% inhibited by the addition of azide or catalase to the incubations indicating that bioactivation was peroxidase-mediated. In contrast, the addition of 1-naphthol to PMA-stimulated neutrophils interfered with superoxide-dependent cytochrome c reduction as well as luminol-dependent chemiluminescence and also resulted in protein binding. Protein binding was only partially inhibited by azide or catalase. The addition of SOD in combination with catalase resulted in a significantly greater inhibition of binding when compared to that of catalase alone. The results of these experiments indicate that phenol and 1-naphthol are converted to reactive metabolites during superoxide generating conditions but by different mechanisms. The formation of reactive metabolites from phenol was almost exclusively peroxidase-mediated whereas the bioactivation of 1-naphthol could occur by two different mechanisms, a peroxidase-dependent and a direct superoxide-dependent mechanism.     

Studies on the active site of rat glutathione S-transferase isoenzyme 4-4. Chemical modification by tetrachloro-1,4-benzoquinone and its glutathione conjugate

The active site of glutathione S-transferase isoenzyme 4-4, purified from rat liver, was studied by chemical modification. Tetrachloro-1,4-benzoquinone, a compound previously shown to inactivate glutathione S-transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro-1,4-benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue. Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro-1,4-benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate. The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro-1,4-benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue. Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro-1,4-benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70-80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S-transferase 4 subunit.     

Cloning of a gene cluster involved in the catabolism of p-nitrophenol by Arthrobacter sp. strain JS443 and characterization of the p-nitrophenol monooxygenase

The npd gene cluster, which encodes the enzymes of a p-nitrophenol catabolic pathway from Arthrobacter sp. strain JS443, was cloned and sequenced. Three genes, npdB, npdA1, and npdA2, were independently expressed in Escherichia coli in order to confirm the identities of their gene products. NpdA2 is a p-nitrophenol monooxygenase belonging to the two-component flavin-diffusible monooxygenase family of reduced flavin-dependent monooxygenases. NpdA1 is an NADH-dependent flavin reductase, and NpdB is a hydroxyquinol 1,2-dioxygenase. The npd gene cluster also includes a putative maleylacetate reductase gene, npdC. In an in vitro assay containing NpdA2, an E. coli lysate transforms p-nitrophenol stoichiometrically to hydroquinone and hydroxyquinol. It was concluded that the p-nitrophenol catabolic pathway in JS443 most likely begins with a two-step transformation of p-nitrophenol to hydroxy-1,4-benzoquinone, catalyzed by NpdA2. Hydroxy-1,4-benzoquinone is reduced to hydroxyquinol, which is degraded through the hydroxyquinol ortho cleavage pathway. The hydroquinone detected in vitro is a dead-end product most likely resulting from chemical or enzymatic reduction of the hypothetical intermediate 1,4-benzoquinone. NpdA2 hydroxylates a broad range of chloro- and nitro-substituted phenols, resorcinols, and catechols. Only p-nitro- or p-chloro-substituted phenols are hydroxylated twice. Other substrates are hydroxylated once, always at a position para to a hydroxyl group.     

Mechanism of combined oxidation of ascorbic acid and hydroquinone in the presence of horseradish peroxidase]

A steady-state kinetics of peroxidase cooxidation of ascorbic acid and hydroquinone catalyzed by horseradish peroxidase was studied. Ascorbic acid and hydroquinone were shown to be oxidized successively, and hydroquinone promoted the oxidation of ascorbic acid. Excess ascorbic acid inhibited peroxidase in the cooxidation of the substrates at pH 5-7. The values of catalytic constants, (kcat, K(m), and Ka) were determined. A possible activation mechanism of the peroxidation of ascorbic acid in the presence of hydroquinone was suggested, and its biological significance was considered.     

Macrophage binding of cells resistant and sensitive to contact-dependent cytotoxicity

We compared macrophage binding and killing of F5b cells to the binding and killing of P815 mastocytoma cells and to several other nontransformed and transformed cell lines. Formalin fixation of elicited or activated macrophages did not affect binding of F5b or 3T3 cells but did abrogate binding of P815 cells. However, formalin fixation abrogated resident macrophage binding of F5b and 3T3 cells. Therefore, depending on the type of macrophage or target cell, formalin fixation may affect binding. Only the binding of P815 cells was dependent upon activation; macrophage binding of target cells F5b and 3T3 was not. Even though macrophages bound F5b and 3T3 cells, macrophages only mediated contact-dependent cytotoxicity against F5b cells. Macrophages did not kill 3T3 cells. Experiments also compared macrophage binding and killing of the uv-light-induced tumor cell lines 1422, 2237, and 2237a46. Only the cell line 2237a46 was susceptible to contact-dependent killing. Both 1422 and 2237 cells were resistant. In contrast, cell lines 2237a46 and 1422 were bound by activated macrophages while 2237 cells were bound poorly.     

Degradation of 2,7-dichlorodibenzo-p-dioxin by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions, the white-rot basidiomycete Phanerochaete chrysosporium degraded 2,7-dichlorodibenzo-p-dioxin (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell-free extracts. The multistep pathway involves the degradation of I and subsequent intermediates by oxidation, reduction, and methylation reactions to yield the key intermediate 1,2,4-trihydroxybenzene (III). In the first step, the oxidative cleavage of the dioxin ring of I, catalyzed by LiP, generates 4-chloro-1,2-benzoquinone (V), 2-hydroxy-1,4-benzoquinone (VIII), and chloride. The intermediate V is then reduced to 1-chloro-3,4-dihydroxybenzene (II), and the latter is methylated to form 1-chloro-3,4-dimethoxybenzene (VI). VI in turn is oxidized by LiP to generate chloride and 2-methoxy-1,4-benzoquinone (VII), which is reduced to 2-methoxy-1,4-dihydroxybenzene (IV). IV is oxidized by either LiP or MnP to generate 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (III). The other aromatic product generated by the initial LiP-catalyzed cleavage of I is 2-hydroxy-1,4-benzoquinone (VIII). This intermediate is also generated during the LiP- or MnP-catalyzed oxidation of the intermediate chlorocatechol (II). VIII is also reduced to 1,2,4-trihydroxybenzene (III). The key intermediate III is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial oxidative cleavage of both C-O-C bonds in I by LiP generates two quinone products, 4-chloro-1,2-benzoquinone (V) and 2-hydroxy-1,4-benzoquinone (VIII). The former is recycled by reduction and methylation reactions to generate an intermediate which is also a substrate for peroxidase-catalyzed oxidation, leading to the removal of a second chlorine atom. This unique pathway results in the removal of both aromatic chlorines before aromatic ring cleavage takes place.     

Isolation and identification of secondary metabolites of a strain ofAspergillus terreus,a common food contaminant

2-Hydroxy 3-methyl 1,4-benzoquinone 5,6 epoxide was identified as secondary metabolite of a strain ofAspergillus terreus, a common contaminant of animal feeds. In addition, the following compounds were also tentatively identified to be produced by this organism: 2-hYdroxy 3-methyl 1,4-benzoquinone; 2-methyl 1,4-benzoquinone 5,6-epoxide; naphthazarin epoxide; and 2-hydroxy 3-methyl 1,4-benzoquinone 5, 6-epoxide.     

Degradation of 2,4-dichlorophenol by the lignin-degrading fungus Phanerochaete chrysosporium.

Under secondary metabolic conditions the white rot basidiomycete Phanerochaete chrysosporium mineralizes 2,4-dichlorophenol (I). The pathway for the degradation of 2,4-dichlorophenol (I) was elucidated by the characterization of fungal metabolites and of oxidation products generated by purified lignin peroxidase and manganese peroxidase. The multistep pathway involves the oxidative dechlorination of 2,4-dichlorophenol (I) to yield 1,2,4,5-tetrahydroxybenzene (VIII). The intermediate 1,2,4,5-tetrahydroxybenzene (VIII) is ring cleaved to produce, after subsequent oxidation, malonic acid. In the first step of the pathway, 2,4-dichlorophenol (I) is oxidized to 2-chloro-1,4-benzoquinone (II) by either manganese peroxidase or lignin peroxidase. 2-Chloro-1,4-benzoquinone (II) is then reduced to 2-chloro-1,4-hydroquinone (III), and the latter is methylated to form the lignin peroxidase substrate 2-chloro-1,4-dimethoxybenzene (IV). 2-Chloro-1,4-dimethoxybenzene (IV) is oxidized by lignin peroxidase to generate 2,5-dimethoxy-1,4-benzoquinone (V), which is reduced to 2,5-dimethoxy-1,4-hydroquinone (VI). 2,5-Dimethoxy-1,4-hydroquinone (VI) is oxidized by either peroxidase to generate 2,5-dihydroxy-1,4-benzoquinone (VII) which is reduced to form the tetrahydroxy intermediate 1,2,4,5-tetrahydroxybenzene (VIII). In this pathway, the substrate is oxidatively dechlorinated by lignin peroxidase or manganese peroxidase in a reaction which produces a p-quinone. The p-quinone intermediate is then recycled by reduction and methylation reactions to regenerate an intermediate which is again a substrate for peroxidase-catalyzed oxidative dechlorination. This unique pathway apparently results in the removal of both chlorine atoms before ring cleavage occurs.     

Co-operation between plasmin and elastase in elastin degradation by intact murine macrophages.

Intact, thioglycollate-stimulated murine macrophages cultured on an insoluble [3H]-elastin substratum progressively hydrolysed the elastin. Cell lysates had little activity. We compared the effect of various proteinase inhibitors on elastinolysis by either live cells or cell-free, elastase-rich conditioned medium. Only known inhibitors of macrophage elastase blocked the activity of elastase-rich cell-conditioned medium whereas inhibitors of cathepsin B also suppressed intact cell activity. Serum proteinase inhibitors blocked cell-derived soluble elastase activity but not intact cell elastolytic activity. We also observed that plasminogen added to the cell cultures markedly increased elastinolysis by live macrophages or cell-free elastase-rich medium. Purified plasmin alone had no measurable effect on native elastin. Additional experiments indicated that the plasmin enhancement was due to elastin-dependent activation of latent macrophage elastase. These results indicate that live macrophage elastinolysis is a co-operative process involving multiple proteinases, especially a cysteine proteinase(s) and elastase. Plasmin may be a physiological activator of latent macrophage elastase.     

Identification of leishmania fructose-1,6-bisphosphate aldolase as a novel activator of host macrophage Src homology 2 domain containing protein tyrosine phosphatase SHP-1

The macrophage protein tyrosine phosphatase-1 SHP-1 has been implicated in the pathogenesis of infection with leishmania. To identify the factors that may interact with SHP-1, Leishmania donovani promastigote lysates were added to a GST-SHP-1 affinity matrix. A 44 kDa specifically bound protein was identified as leishmania fructose-1,6-bisphosphate aldolase (aldolase). Purified leishmania aldolase bound to SHP-1 indicating that the interaction was direct. In contrast, purified mammalian aldolase did not bind to SHP-1. Consistent with this, leishmania aldolase activated SHP-1 in vitro, whereas mammalian aldolase did not. The presence of leishmania aldolase in the cytosolic fractions prepared from infected macrophages indicated that leishmania aldolase is exported from phagolysosomes in infected cells where it can target host cytosolic proteins. In fact, co-immunoprecipitation showed association of leishmania aldolase with SHP-1. Moreover, leishmania aldolase-expressing macrophages showed the deactivated phenotype of leishmania infected cells as judged by much reduced inability to induce expression of nitric-oxide synthase in response to interferon-γ treatment. Collectively, these data show that leishmania aldolase is a novel SHP-1 binding and activating protein that contributes to macrophage dysfunction.     

Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro

Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation ara cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbonyl-14C-labeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this compound is activated by microsomal enzymes. However, only about 5% of this metabolite can be accounted for as protein-bound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole.     

IFN-gamma reduces specific binding of tumor necrosis factor on murine macrophages.

Because IFN-gamma is the main cytokine activating macrophages and TNF cooperates in this activation, we assessed TNF binding capacity during the course of murine macrophage activation by IFN-gamma. TNF binding to elicited macrophages increased with time, was maximal by 8 h of culture, and required de novo protein synthesis. 125I-TNF bound to about 40,000 sites/cell with a Kd of 1 x 10(-9) M. Cross-linking experiments performed with a bifunctional cross-linking agent revealed a specific band with a m.w. of 94,000. Preincubation of macrophages with IFN-gamma prevented the binding of TNF to receptors. This effect was dose-dependent and maximal at 100 U/ml. IFN-gamma also reduced specific TNF binding to preexisting receptors (50% inhibition in 3 h), but IFN-gamma did not change the internalization rate of TNF. These studies showed that the number of TNF receptors increased on macrophages vs maturation in culture and was negatively controlled by IFN-gamma.