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PubMed1988

Effect of phenylhydrazine on red blood cell metabolism.

In addition to the well known effect of phenylhydrazine on red blood cells (methaemoglobin and Heinz body formation, autologous IgG binding, lipid peroxidation, etc.) an increased glucose utilization was observed. Measurement of 14CO2 formation from [1-14C]-glucose showed a maximum value at 2mM phenylhydrazine followed by a progressive inhibition on increasing the drug concentration to 16 mM. Concomitantly we found a reduction in the reduced glutathione concentration but not a corresponding increase in the level of oxidized glutathione. Phenylhydrazine also causes ATP depletion. The ATP is in part dephosphorylated to ADP and AMP and in part converted to inosine monophosphate and hypoxanthine. Measurement of the cell content of reduced and oxidized pyridine nucleotides was also performed and showed a progressive increase in the reduced forms of these coenzymes. Thus phenylhydrazine promotes cellular ATP depletion followed by adenine nucleotide catabolism that is not efficiently counteracted by an increase in glucose utilization. The relevance of these data to the mechanism of phenylhydrazine-induced anemia is discussed.

Adenine Nucleotides↗

PubMed1995

Reaction mechanism for the inactivation of the quinoprotein methylamine dehydrogenase by phenylhydrazine.

Phenylhydrazine has previously been shown to be an irreversible inactivator of the tryptophan tryptophylquinone (TTQ) enzyme methylamine dehydrogenase [Davidson, V.L. and Jones, L.H. (1992) Biochim. Biophys. Acta 1121, 104-110]. Structure-reactivity correlations have been performed to elucidate the mechanism by which this inactivation occurs. The reactions of a series of p-substituted phenylhydrazines with methylamine dehydrogenase were examined. Correlation with electronic substituent effects was observed. A Hammett plot of the second order inactivation rate constants versus sigma p exhibited a positive slope. Plots of these rate constants against substituent constants which reflected either resonance or field/inductive parameters for each p-substituent indicated that the rate was primarily influenced by resonance electronic effects. A Brønsted plot of the inactivation rate constant against pKa of each substituted phenylhydrazine yielded a beta-value (slope) of 0.7. Based upon these results, a reaction mechanism is proposed for the inactivation of methylamine dehydrogenase by phenylhydrazines, and a structure is proposed for the putative transition state for the rate-limiting step in the overall processes of binding and adduct formation by phenylhydrazine. The relevance of these results to the process of imine formation between substrate amines and TTQ during the normal catalytic process is also discussed.

Indolequinones↗

PubMed1989

Age-related and phenylhydrazine-induced activation of the membrane-associated cathepsin E in human erythrocytes.

We examined the effects of cell aging and phenylhydrazine-induced oxidant damage on erythrocyte cathepsin E, which is present as a latent, membrane-associated enzyme in normal human erythrocytes. When young erythrocytes isolated from human mature erythrocytes by Percoll density gradient centrifugation were aged in vitro, the membrane-associated cathepsin E was progressively released from the membrane as an active enzyme. During the cell aging up to 100 h, about 40% of the membrane-associated enzyme was activated and solubilized. When phenylhydrazine was incubated with the erythrocytes, it also caused the activation and solubilization of cathepsin E in a dose-dependent and time-dependent manner. Exposure of erythrocytes to 2.5 mM phenylhydrazine for up to 2 h led to about 40% activation of the membrane-associated enzyme. Both aging and phenylhydrazine-treatment were accompanied with an increase in the association of the cytosolic proteins, primarily hemoglobin, with the membrane, which occurred prior to the release of cathepsin E from the membrane. A similar activation for the membrane-associated enzyme was observed with in vitro-aged hemoglobin-free membrane ghosts. Thus, the primary mechanism for activation of cathepsin E in the intact cells seems to be through lesion of the membrane framework that results from increased binding of hemoglobin to the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting employing polyclonal IgG antibodies for human spectrin and band 3 revealed that breakdown of the membrane proteins was enhanced in both aged and phenylhydrazine-treated cells. The relation between the cathepsin E activation and the membrane protein breakdown is discussed.

Cathepsin E↗

PubMed1985

Reactions of hemoglobin with phenylhydrazine: a review of selected aspects.

It is well known that phenylhydrazine induces hemolytic anemia. This is thought to result from the reaction of phenylhydrazine with hemoglobin. The accompanying oxidation of phenylhydrazine leads to the formation of a number of products, including benzene, nitrogen, hydrogen peroxide, superoxide anion and the phenyl radical. The products formed depend critically on the conditions of the experiment, especially the amount of oxygen present. It is now known that oxyhemoglobin and myoglobin react with phenylhydrazine to yield a derivative of hemoglobin containing N-phenylprotoporphyrin in which the heme group is modified. The recent identification of sigma-phenyliron(III) porphyrins in phenylhydrazine-modified metmyoglobin has aided elucidation of the mechanism of hemoglobin modification. Mechanistic schemes are proposed to account for product formation.

Anemia, Hemolytic↗

PubMed1990

Active site labeling of dopamine beta-hydroxylase by two mechanism-based inhibitors: 6-hydroxybenzofuran and phenylhydrazine.

6-Hydroxybenzofuran and phenylhydrazine are mechanism-based inhibitors of dopamine beta-hydroxylase (D beta H; EC 1.14.17.1). We report here the isolation and characterization of radiolabeled peptides obtained after inactivation of D beta H with [3H]6-hydroxybenzofuran and [14C]phenylhydrazine followed by digestion with Staphylococcus aureus V8 protease. Inactivation of D beta H with [3H]6-hydroxybenzofuran gave only one labeled peptide, whereas inactivation with [14C]phenylhydrazine gave several labeled peptides. Each inhibitor labeled a unique tyrosine in the enzyme corresponding to Tyr477 in the primary sequence of the bovine enzyme (Robertson, J. G., Desai, P. R., Kumar, A., Farrington, G. K., Fitzpatrick, P. F., and Villafranca, J. J. (1990) J. Biol. Chem. 265, 1029-1035). In addition, [14C]phenylhydrazine also labeled a unique histidine (His249) as well as several other peptides. Examination of the complete peptide profile obtained by high pressure liquid chromatography analysis also revealed the presence of a modified but nonradioactive peptide. This peptide was isolated and sequenced and was identical whether the enzyme was inactivated by 6-hydroxybenzofuran or phenylhydrazine. An arginine at position 503 was missing from the sequence cycle performed by Edman degradation of the modified peptide, but arginine was present in the identical peptide isolated from native dopamine beta-hydroxylase. These data are analyzed based on an inactivation mechanism involving formation of enzyme bound radicals (Fitzpatrick, P. F., and Villafranca, J. J. (1986) J. Biol. Chem. 261, 4510-4518) interacting with active site amino acids that may have a role in substrate binding and binding of the copper ions at the active site.

Amino Acid Sequence↗

PubMed1988

In vivo rat hemoglobin thiyl free radical formation following phenylhydrazine administration.

The reaction of oxyhemoglobin with phenylhydrazine has received considerable attention for many decades. The basis for this interest stems from the ability of phenylhydrazine and hydrazine-based drugs to induce hemolytic anemia. Considerable evidence obtained from in vitro ESR experiments implicates free radicals in the events leading to red blood cell hemolysis. However, until this report, no corroborating ESR evidence for in vivo free radical formation has been presented. We have successfully employed ESR to detect the formation of a radical adduct in the blood of rats which received an intragastric dose of phenylhydrazine followed by an intraperitoneal injection of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). An immobilized radical adduct was detected by ESR when phenylhydrazine was administered in a dosage comparable to that prescribed for currently employed hydrazine-based drugs. We were also able to detect this immobilized DMPO adduct when hydrazine was employed in place of phenylhydrazine in the rat studies. The results of a series of experiments led us to ascribe this DMPO radical adduct to the trapping of a hemoglobin-derived thiyl free radical.

Animals↗

PubMed1985

Role of free radical scavengers on phenylhydrazine induced hemolysis in rat.

The mechanism of the phenylhydrazine induced oxidative hemolysis was studied on the point of role of the free radical scavengers in rats. Phenylhydrazine resulted in the degradation of hemoglobin and the lipid peroxidation of the erythrocyte membrane. Otherwise, the elevation of coenzyme Q9, endogenous CoQ in rats, levels in plasma was observed against the phenylhydrazine induced oxidative stress. Supplementation of coenzyme Q10, exogenous CoQ in rats, inhibited the phenylhydrazine induced hemolysis according to the suppression of both the degradation of hemoglobin and the lipid peroxidation of the erythrocyte membrane. These results suggest that free radical scavengers such as coenzyme Q9 and coenzyme Q10 have important roles on the phenylhydrazine induced hemolysis in rats.

Animals↗

PubMed1983

Inactivation of catalase by phenylhydrazine. Formation of a stable aryl-iron heme complex.

Catalase promotes the H2O2-dependent oxidation of phenylhydrazine to benzene but simultaneously is subject to a pseudo-first order inactivation process. Each inactivation event is subtended by catalytic turnover of three molecules of phenylhydrazine and 52 molecules of H2O2. The dimethyl ester of N-phenylprotoporphyrin IX is extracted with acidic methanol from the inactivated enzyme, but the prosthetic heme with a phenyl sigma-bonded to the iron atom is obtained by gentle extraction with 2-butanone. The absolute chirality of N-ethylprotoporphyrin IX isolated from catalase inactivated with ethylhydrazine confirms that the prosthetic heme has the same chiral orientation in the active site as it does in hemoglobin. The known inactivation of methemoglobin by phenylhydrazine is shown to depend on H2O2 but not oxygen. The results demonstrate that the H2O2-dependent oxidation of phenylhydrazine by catalase and other hemoproteins results in sigma-coordination of a phenyl residue to the prosthetic heme iron. This process may play a role not only in phenylhydrazine-mediated erythrocyte lysis but also in the activation of guanylate cyclase.

Animals↗

PubMed1989

Differential location of hemopoietic colonies within liver acini of postnatal and phenylhydrazine-treated adult mice.

We have measured the location of embryonic and adult hemopoietic foci in the liver tissue of postnatal and adult phenylhydrazine-treated mice. Differentiation of acinar domains in liver tissue was made possible by carrying out succinate dehydrogenase histochemical reactions on liver cryostat sections. To determine the position of hemopoietic foci within the lobular gradient of the hepatocyte succinate dehydrogenase activity, this enzyme was measured in hepatocytes surrounding both portal and central veins and hemopoietic foci. Then, assuming the periportal succinate dehydrogenase activity value to be 1.00 +/- 0.2, succinate dehydrogenase activity around postnatal hemopoietic foci was 0.65 +/- 0.19, around phenylhydrazine-induced hemopoietic foci 0.83 +/- 0.24 and around central veins 0.44 +/- 0.11. Scaling the portal to central vein distance and taking 1 as the portal vein point and 0 as the central vein point, the relative position of hemopoietic foci, indirectly calculated from succinate dehydrogenase activity values, was 0.35 +/- 0.13 in postnatal livers and 0.73 +/- 0.12 in phenylhydrazine-treated adult livers. Hemopoietic foci frequencies varied according to both the origin and the liver acinar domain: in postnatal liver acini, it was 37.1% in zone 1, 22.8% in zone 2 and 40% in zone 3; in phenylhydrazine-treated adult acini, it was 89.4% in zone 1 and 10.6% in zone 2. Postnatal hemopoietic foci mainly occurred extrasinusoidally, between hepatocytes and reticular-like cells, whereas adult hemopoietic foci were mostly intrasinusoidal and closely associated to macrophage-like cells. Adult hemopoietic colonies

Age Factors↗

PubMed1982

Development of the embryonic chick phagocytic system: intraembryonic erythrophagocytosis induced by phenylhydrazine.

The intraembryonic reticuloendothelial response to phenylhydrazine-induced hemolytic anemia was studied embryonic chicks (days 13-16) by light and electron microscopy and histochemical and biochemical assays for acid phosphatase. Phenylhydrazine was given on day 13 and tissue taken at 2, 5, and 10 h and at 1, 2, and 3 days after injections. The response varied in the three major reticuloendothelial organs. The spleen first demonstrated an increase in erythrophagocytosis that was accompanied by increased acid phosphatase levels. Erythrophagocytosis occurred primarily in the red pulp resulting in increased numbers of macrophages, increased to enlarge the spleens. By 2 days after phenylhydrazine injection, greatly enlarged macrophages began to migrate into the venous system, where some erythrophagocytosis continued to occur. The liver was also a major erythroclastic organ in which Kupffer cells became increasingly erythrophagocytic. However, erythrophagocytosis began later than in the spleen, and as measured by acid phosphatase levels, the liver was not as effective in removing damaged erythroid cells. Marrow erythrophagocytosis was only slightly enhanced; however, the marrow responded by increasing its production of red blood cells. Thus, the intraembryonic reticuloendothelial organs of the embryonic chick responded to phenylhydrazine-induced hemolytic anemia in much the same manner as might be expected of the adult bird.

Acid Phosphatase↗

PubMed1987

Isolation of N-phenylprotoporphyrin IX from the red cells and spleen of the phenylhydrazine-treated rat.

Blood and spleens of phenylhydrazine-injected rats were treated with a solution of acidic methanol and zinc ion to isolate a green pigment. The pigment was resolved into two, I and II, by thin-layer chromatography. Pigment I was a mixture of two isomers of zinc complex of esterified N-phenylprotoporphyrin IX, in which vinyl-substituted pyrrole rings A and B were phenylated; and pigment II was a mixture of two isomers of the porphyrin complex with the N-phenyl group on propionic acid-substituted rings C and D. These pigments were also chemically prepared from the reaction of phenylhydrazine with oxyhemoglobin, independently characterized, and used to confirm the structures of the biological pigments. Determination revealed that the total amount of pigments found in the blood and spleen at 24 h after injection of phenylhydrazine corresponds to about 0.4% of the injected phenylhydrazine.

Animals↗

PubMed1990

Inactivation of lignin peroxidase by phenylhydrazine and sodium azide.

Lignin peroxidase (LiP) is rapidly inactivated in a concentration-dependent manner by H2O2 and either phenylhydrazine or sodium azide. Full inactivation of isozyme 2b (H8) requires approximately 50 eq of phenylhydrazine or 80 eq of sodium azide. Anaerobic incubation of isozyme 2b with [14C]phenylhydrazine and H2O2 results in 77% loss of catalytic activity and covalent binding of 0.45 mol radiolabel/mol of enzyme. Comparable but not identical results are obtained with an isozyme mixture. A lag period is observed before the peroxidative activity can be measured when an aliquot of an incubation with sodium azide is diluted into the mixture used to assay residual catalytic activity. This lag is associated with reversible accumulation of a catalytically inert species with a Compound III-like spectrum. No meso-phenyl, iron-phenyl, or N-phenyl adducts are formed with phenylhydrazine but a low yield of what appears to be delta-meso-azidoheme is obtained with sodium azide. LiP is thus less susceptible to meso heme additions and more susceptible to oxidative heme degradation than horseradish peroxidase. The data suggest that the active of LiP resembles the closed structure of horseradish peroxidase more than it does the open structure of the globins, catalase, chloroperoxidase, or cytochrome P450.

Azides↗

PubMed1990

Phenylhydrazine mediated degradation of bovine serum albumin and membrane proteins of human erythrocytes.

Phenylhydrazine in solution has been shown to produce hydroxyl radicals, as measured by 2-deoxyribose degradation assay. In vitro incubation of bovine serum albumin with phenylhydrazine leads to extensive degradation of the former which, however, is not inhibited by hydroxyl-radical scavengers like mannitol, Tris or n-butanol. Metal chelators like EDTA, however, inhibits the breakdown of BSA. Erythrocyte ghosts incubated in vitro with phenylhydrazine also show extensive loss of membrane cytoskeletal proteins, inhibited by 5 mM EDTA but not by mannitol. In both the occasions a hydroxyl-radical mediated damage to protein takes place by a 'site-specific mechanism'. Further, such a damage to erythrocyte membrane proteins by phenylhydrazine may be related to well known action of this compound in producing accelerated aging of erythrocytes.

Cytoskeletal Proteins↗

PubMed1991

Oxidative damage by phenylhydrazine diminishes erythrocyte anion transport.

Human erythrocytes were exposed to oxidative stress by treatment with the slowly hemolytic drug phenylhydrazine. Phenylhydrazine has been previously shown to trigger the production of toxic oxygen metabolites including O-2 and H2O2 and the formation of Heinz bodies. The concentration-dependent formation of Heinz bodies was confirmed using optical microscopy. Heinz body formation was accompanied by surface protuberances as shown by scanning electron microscopy. The formation of Heinz bodies was accompanied by inhibition of anion translocation. Anion translocation was measured using the anionic fluorescent substrate analog N-(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-1,3-diazole (NBD-taurine). The efflux of NBD-taurine was measured by continuous monitoring of transport by fluorescence (CMTF). The mean value of the kinetic rate constant for transport, k, was found to be -0.090 +/- 0.017 min-1. Phenylhydrazine was found to decrease k to less than one-half of control values in a dose-dependent fashion. The disruption of anion translocation may be related to the oxidative effects of phenylhydrazine and to the generation of Heinz bodies, which bind to the N-terminal domain of band 3.

Adult↗

PubMed1984

Reaction of phenylhydrazine with erythrocytes. Cross-linking of spectrin by disulfide exchange with oxidized hemoglobin.

Phenylhydrazine causes deleterious oxidations of components of erythrocytes. These reactions and their effects on the mechanical properties of rabbit erythrocytes are investigated to provide insight into the mechanisms leading to destruction of oxidatively damaged erythrocytes. After 1 hr of incubation with phenylhydrazine, precipitated denatured protein (Heinz body protein) amounts to 25-30% of membrane protein, but deformability of erythrocytes as measured by filtrability is unchanged. After 4 hr of incubation filtrability drops sharply. Polymerization of spectrin and covalent binding of hemoglobin to spectrin, but no peroxidation of membrane lipids is observed. Precipitated protein amounts to 85-95% of membrane protein. It contains Fe, porphyrin and globin peptide in the proportion 1:1:1. Heinz body protein precipitated when hemoglobin is incubated under similar conditions has 90% of its sulfhydryl groups oxidized and no other amino acids than cysteine are destroyed. Addition of this Heinz body protein to erythrocyte ghosts causes polymerization of spectrin. Incubation of tetrathionate, a specific cross-linking agent, causes filtrability to drop sharply after about 80 min. This effect is similar to that observed after 4 hr incubation with phenylhydrazine, and is accompanied by polymerization of spectrin and band 3. The results indicate that cross-linking of membrane proteins by disulfide exchange with precipitated hemoglobin may play a major role in decreasing deformability during incubation with phenylhydrazine.

Amino Acids↗

PubMed1982

Industrial hygiene air monitoring of phenylhydrazine.

Two gas chromatographic (GC) methods for the analysis of phenylhydrazine have been examined with phenyl- and rho-chlorophenylhydrazine and a new method using thin-layer chromatography (TLC) was developed. The NIOSH GC method (2-furalphenylhydrazone derivatization), which is classified as "proposed", was found to yield two derivatization peaks, which could only be separated with capillary columns. The other GC method (acetone phenylhydrazone derivatization) gave a single peak, however, of limited stability. In the newly developed TLC method the fluorescamine derivative of phenylhydrazine is separated and detected either visually or with a TLC-fluorescence scanner after fluorescence enhancement with paraffin wax. With ordinary TLC techniques the quantification of phenylhydrazine was possible down to 200 pg per spot. The sensitivity of the TLC method is generally sufficient for the requirements of industrial hygiene air monitoring of phenylhydrazine.

Air↗

PubMed1982

Mechanism of initial reaction of phenylhydrazine with oxyhemoglobin and effect of ring substitutions on the biomolecular rate constant of this reaction.

Phenylhydrazine in the presence of oxygen causes the oxidative denaturation of hemoglobin. The initial step in this process is a bimolecular reaction, probably a two-electron transfer from phenylhydrazine to oxyhemoglobin. The product of this reaction is neither methemoglobin nor deoxyhemoglobin. Superoxide dismutase and catalase eliminate side reactions that increase the apparent rate of this reaction as measured spectrophotometrically at 577 nm; scavengers for the hydroxyl radical and singlet oxygen do not affect this rate either in the presence or in the absence of these enzymes. Halogen atoms and alkyl groups decrease the rate when ortho and increase the rate when meta or para to the hydrazino group. Chlorine atoms at both ortho positions or the carboxylate group at the ortho or the para position block the reaction. In the presence of phenylhydrazine under air, methemoglobin is converted to the same complex as that produced when phenyldiazene is added to methemoglobin anaerobically. Under N2 or CO, phenylhydrazine reduces methemoglobin to deoxyhemoglobin or carbonmonoxyhemoglobin.

Catalase↗

PubMed1996

Probing the aromatic-donor-binding site of horseradish peroxidase using site-directed mutagenesis and the suicide substrate phenylhydrazine.

The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-access-channel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (delta-meso phenyl) and C18-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03% of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkable constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why phenylhydrazine-treated C. cinereus peroxidase was more resistant to haem modification and phenyl-radical-based inactivation than HRP-C.

Binding Sites↗