Peroxidase-catalysed binding of [U-14C]phenol to DNA

14C-Phenol binds irreversibly to calf-thymus DNA in the presence of horseradish peroxidase and hydrogen peroxide, approximately 65% of the added phenol was bound to DNA. Binding was maximal at an equimolar concentration of hydrogen peroxide. Binding also occurred to homopolyribonucleotides polyadenylic acid, polyguanylic acid, polycytidylic acid and polyuridylic acid, and suggests that binding is relatively non-specific with respect to the nucleotide bases. p,p'-Biphenol, p,p'-biphenoquinone, o,o'-biphenol and two unidentified products were formed by the oxidation of phenol, in the presence and in absence of DNA. DNA accelerated phenol oxidation four fold and prevented the polymerization of oxidized phenol products, but was found to have no effect on the range of ethyl acetate-extractable products. Phenol accelerated the metabolism of o,o'-biphenol but had no effect on p,p'-biphenol metabolism. The mechanism of phenol activation is not clear, but p,p'-biphenoquinone binds to protein and not to DNA. DNA binding was prevented by glutathione, N-acetyl-cysteine and ascorbate, and the mechanism was shown to involve reduction of the activated phenol intermediates and the formation of conjugates with glutathione and N-acetyl-cysteine. DNA binding was not inhibited by lysine and proline.     

Peroxidase-catalysed binding of [U-14C]phenol to DNA

1. ¹⁴C-Phenol binds irreversibly to calf-thymus DNA in the presence of horseradish peroxidase and hydrogen peroxide, approximately 65% of the added phenol was bound to DNA. Binding was maximal at an equimolar concentration of hydrogen peroxide. Binding also occurred to homopolyribonucleotides polyadenylic acid, polyguanylic acid, polycytidylic acid and polyuridylic acid, and suggests that binding is relatively non-specific with respect to the nucleotide bases.

2. p,p'-Biphenol, p,p'-biphenoquinone, o,o'-biphenol and two unidentified products were formed by the oxidation of phenol, in the presence and in absence of DNA. DNA accelerated phenol oxidation four fold and prevented the polymerization of oxidized phenol products, but was found to have no effect on the range of ethyl acetate-extractable products. Phenol accelerated the metabolism of o,o'-biphenol but had no effect on p,p'-biphenol metabolism. The mechanism of phenol activation is not clear, but p,p'-biphenoquinone binds to protein and not to DNA.

3. DNA binding was prevented by glutathione, N-acetyl-cysteine and ascorbate, and the mechanism was shown to involve reduction of the activated phenol intermediates and the formation of conjugates with glutathione and N-acetyl-cysteine. DNA binding was not inhibited by lysine and proline.     

Phenol oxidation product(s), formed by a peroxidase reaction,that bind to DNA

1. Phenol oxidation by horseradish peroxidase/H₂O₂ initially results in p,p'-biphenol and o,o'-biphenol formation and subsequently results in polymer formation. o,o'-Biphenol is the major product formed, but it is rapidly oxidized to the polymer, particularly in the presence of phenol. p,p'-Biphenol is very rapidly oxidized to p,p'-biphenoquinone which can also be involved in polymer formation.

2. Extensive binding of ¹⁴C-phenol oxidation products to DNA occurs if the DNA is present in the reaction mixture. However, enzymic hydrolysis of DNA releases the bound polymers. p,p'-Biphenol, however, did not bind to DNA following peroxidase-catalysed oxidation, but o,o'-biphenol readily binds to DNA following peroxidase-catalysed oxidation. Enzymic hydrolysis of the oxidized o,o'-biphenol-bound DNA also resulted in the release of the polymers.     

Phenol-induced stimulation of hydroquinone bioactivation in mouse bone marrow in vivo: possible implications in benzene myelotoxicity

The coadministration of phenol and hydroquinone has been shown to produce myelotoxicity in mice similar to that observed following benzene exposure. One explanation of this phenomenon may be that phenol enhances the peroxidase-dependent metabolic activation of hydroquinone in the mouse bone marrow. Here we report that radiolabeled [14C]hydroquinone and [14C]phenol bind covalently to tissue macromolecules of blood, bone marrow, liver and kidney, when administered intraperitoneally to the mouse in vivo. Substantially more radiolabeled hydroquinone was covalently bound 18 h after administration as compared with that bound after 4 h. Phenol, when administered together with [14C]hydroquinone, significantly stimulated the covalent binding of [14C]hydroquinone oxidation products to blood (P less than 0.001) and bone marrow (P less than 0.05) macromolecules, but had no significant effect on covalent binding of [14C]hydroquinone oxidation products to liver and kidney macromolecules (P greater than 0.05). Catechol, on the other hand, had no effect on the binding of [14C]hydroquinone oxidation products in either bone marrow, kidney or liver (P greater than 0.05). When hydroquinone was administered together with [14C]phenol, a stimulation of the covalent binding of phenol oxidation products to bone marrow macromolecules also occurred (P less than 0.05). In addition, hydroquinone co-administration increased the covalent binding of [14C]phenol oxidation products in kidney and blood (P less than 0.05), but significantly decreased the covalent binding in liver (P less than 0.05). These results suggest that altered pharmacokinetics play a major role in the hydroquinone-dependent stimulation of covalent binding of [14C]phenol oxidation products to extrahepatic tissue macromolecules in vivo. The mechanism underlying the phenol-induced stimulation of binding of [14C]hydroquinone by phenol in blood and bone marrow remains unclear, but stimulation of peroxidase-mediated hydroquinone metabolism may be responsible. The latter may therefore play an important role in benzene-induced myelotoxicity.     

An interaction of benzene metabolites reproduces the myelotoxicity observed with benzene exposure

Benzene-induced myelotoxicity can be reproduced by the coadministration of two principal metabolites, phenol and hydroquinone. Coadministration of phenol (75 mg/kg) and hydroquinone (25-75 mg/kg) twice daily to B6C3F1 mice for 12 days resulted in a significant loss in bone marrow cellularity in a manner exhibiting a dose-response. One explanation for this potentiation is that phenol stimulates the peroxidase-dependent metabolism of hydroquinone. Addition of phenol to incubations containing horseradish peroxidase, H2O2, and hydroquinone resulted in a stimulation of both hydroquinone removal and benzoquinone formation. Stimulation occurred with phenol as low as 100 microM and with very low concentrations of horseradish peroxidase. When boiled rat liver protein was added to identical incubations containing [14C]hydroquinone, the level of radioactivity recovered as protein bound increased by 37% when phenol was added. Similar results were observed when [14C]hydroquinone was incubated in the presence of activated human leukocytes. Hydroquinone binding was increased by approximately 70% in the presence of phenol. Phenol-induced stimulation of hydroquinone metabolism and benzoquinone formation represents a likely explanation for the bone marrow suppression associated with benzene toxicity.     

Lack of evidence for a hepatic peroxisome proliferator receptor and an explanation for the binding of hypolipidaemic drugs to liver homogenates

The existence of a postulated hepatic receptor responsible for the peroxisomal proliferation induced in rodents by hypolipidaemic drugs has been investigated. [3H]-nafenopin and [3H]-ciprofibrate were used as labelled ligands and two competitive binding assays, using either a charcoal-dextran or a hydroxylapatite method, were developed to investigate potential binding. In both assay systems, specific displaceable binding of either nafenopin or ciprofibrate to whole homogenate, microsomal and cytosolic fractions of rat liver could not be detected in a variety of buffer systems. A positive control of ligand binding to bovine serum albumin indicated the validity of the binding assays used. In addition, both nafenopin and ciprofibrate exhibited displaceable binding to serum albumin using the hydroxylapatite binding assay and a Scatchard analysis of the binding of [3H]-nafenopin to fatty acid free rat serum albumin yielded a dissociation constant of 5.2 x 10(-7) M and 86 pmol of ligand bound per mg protein. Taken collectively, our data strongly argues against the existence of a specific hepatic peroxisome proliferation receptor and indicates that the peroxisome proliferating hypolipidaemic drugs bind to serum albumin and possibly to other cellular proteins not involved in the activation of genes necessary for peroxisome proliferation.     

The binding of intravenous and oral biliary contrast agents to human and bovine serum albumin

Summary The binding of two homologous series of oral and intravenous biliary contrast agents to human and bovine serum albumin was investigated using the gel filtration technique. All intravenous compounds are bound to human serum albumin via one high affinity and several low affinity binding sites. Within the concentration range investigated, about 3–5 high affinity binding sites for the oral compounds were found on human serum albumin. In general, the intravenous compounds have a greater affinity for human serum albumin than the oral compounds. No significant differences were found for the binding of the oral compounds to human or bovine serum albumin, while the intravenous compounds have a higher affinity for bovine than for human serum albumin. The significance of the plasma protein binding of the biliary contrast agents for the hepatic uptake is discussed.     

Harding-passey mouse-melanoma tyrosinase inactivation by reaction products and activation by L-epinephrine

1. Harding-Passey mouse-melanoma tyrosinase (EC 1.14.18.1) is inhibited during L-3,4-dihydroxyphenylalanine oxidation by reaction products. L-3,4-dihydroxyphenyl 3-[14C]alanine oxidation products bind to the enzyme, as demonstrated by gel electrophoresis and radioactivity measurements. 2. The enzyme interacts with indoles and oxidizes dopamine and norepinephrine. 3. L-epinephrine activates tyrosinase at non hormonal concentrations and bovine serum albumin protects the enzyme from auto-inhibition. 4. The inhibition of the Harding-Passey mouse-melanoma tyrosinase, during substrate oxidation, is very similar to that of mushroom enzyme.     

Aspirin binding and the effect of albumin on spontaneous and enzyme-catalysed hydrolysis

A method of measuring the binding of aspirin to albumin without the interference of hydrolysis was developed. At concentrations of 10 mg litre-1, aspirin is about 85% bound to bovine serum albumin (4 g %), whereas its hydrolysis product, salicylic acid, is 95% bound. Salicylic acid was shown to displace aspirin from albumin binding sites. Both salicylic acid and aspirin bind more strongly to bovine serum albumin than to human serum albumin at protein concentrations of 4 g %. Protein binding protected aspirin against spontaneous hydrolysis although protein-bound aspirin still hydrolysed at a finite rate. In contrast, albumin enhanced the enzyme-catalysed hydrolysis of aspirin. By using a simple model, the rate constants for the individual processes contributing to the overall hydrolysis rate constant in the presence of albumin and esterase are calculated.     

Effect of ascorbate on covalent binding of benzene and phenol metabolites to isolated tissue preparations

[14C]Phenol and [14C]benzene are metabolized in the presence of NADPH and hepatic microsomes isolated from phenobarbital- or benzene-pretreated or untreated guinea pigs to intermediates capable of covalently binding to microsomal protein. When 1 mM ascorbate was included in the incubation mixture containing benzene as the substrate, covalent binding was inhibited by 55%. Increasing the ascorbate concentration to 5 mM inhibited binding by only an additional 17%. In contrast, when phenol was used as the substrate, 1 mM ascorbate inhibited binding by 95%. When DT-diaphorase was included in the incubation mixture containing benzene as the substrate, binding was inhibited by only 18%. This degree of inhibition is in contrast to 70% inhibition with phenol. These results indicate that different metabolites are responsible for a portion of the covalent binding depending upon the substrate employed. GSH inhibited covalent binding greater than 95% with either substrate. The metabolism of phenol to hydroquinone was unaffected by the addition of ascorbate or GSH. The metabolism of benzene to phenol was unaffected by the addition of GSH; however, the addition of ascorbate decreased the formation of phenol by 35%. Tissue ascorbate could be modulated by placing guinea pigs on different dietary intakes of ascorbate. Bone marrow ascorbate concentrations could be modulated 10-fold without any significant change in the GSH concentrations. Bone marrow isolated from guinea pigs on different dietary intakes of ascorbate were incubated with H2O2 and phenol. Bone marrow with low ascorbate concentrations displayed 4-fold more covalent binding of phenol equivalents than those with high ascorbate concentrations. This is an example of how the dietary intake of ascorbate can result in a differential response to a potentially toxic event in vitro.     

Interaction of substituted phenoxazine chemosensitizers with bovine serum albumin

The binding of 10-[3'-[N-bis(hydroxyethyl)amino]propyl]phenoxazine [BPP], 10-[3'-[N-bis(hydroxyethyl)amino]propyl]-2-chlorophenoxazine [BPCP], 10-[3'-[N-bis-(hydroxyethyl)amino]propyl]-2-trifluoromethylphenoxazin e [BPFP], 10-(3'-N-pyrrolidino propyl)-2-chlorophenoxazine [PPCP] or 10-(3'-N-pyrrolidinopropyl)-2-trifluoromethylphenoxazine [PPFP] to bovine serum albumin (BSA) has been measured by gel filtration and equilibrium dialysis methods. The binding of these modulators to bovine serum albumin based on dialysis experiments has been characterized by the following parameters: percentage (beta) of bound drug, the association constant 'K1', the apparent binding constant 'k' and the free energy deltaFdegrees. The binding of phenoxazine derivatives to bovine serum albumin is correlated with their octanol-water partition coefficient, log10P. In addition, the displacing activity of hydroxyzine and acetylsalicylic acid on the binding of phenoxazines to albumin has been studied. The results of the displacing experiments showed that the phenoxazine benzene rings and the tertiary amines attached to the side chain of the phenoxazine moiety are bound to a hydrophobic area on the albumin molecule.     

Comparative investigations of the binding of perazine dimalonate (PDM) to human and bovine serum albumin (author's transl)]

Comparative Investigations of the Binding of Perazine Dimalonate (PDM) to Human and Bovine Serum Albumin The unbound portion of perazine dimalonate (PDM) in buffered solutions of human and bovine serum albumin (pH 7.4) was determined by means of gel filtration and ultrafiltration. Both procedures were optimized by applying techniques which are normaly used in high-pressure liquid chromatography. The binding of the drug to serum albumin was characterized by determination of the overall binding constant (K₁), the apparent binding constant (k⁺), the number of binding sites per molecule of albumin (n) and the free reaction energy (ΔF°). It was found that less PDM is bound by bovine serum albumin than by human serum albumin.     

Aspirin binding and the effect of albumin on spontaneous and enzyme-catalysed hydrolysis

A method of measuring the binding of aspirin to albumin without the interference of hydrolysis was developed. At concentrations of 10 mg litre^?1, aspirin is about 85% bound to bovine serum albumin (4 g%), whereas its hydrolysis product, salicylic acid, is 95% bound. Salicylic acid was shown to displace aspirin from albumin binding sites. Both salicylic acid and aspirin bind more strongly to bovine serum albumin than to human serum albumin at protein concentrations of 4 g%. Protein binding protected aspirin against spontaneous hydrolysis although protein-bound aspirin still hydrolysed at a finite rate. In contrast, albumin enhanced the enzyme-catalysed hydrolysis of aspirin. By using a simple model, the rate constants for the individual processes contributing to the overall hydrolysis rate constant in the presence of albumin and esterase are calculated.     

Covalent binding of food carcinogens MeIQx, MeIQ and IQ to DNA and protein in microsomal incubations and isolated rat hepatocytes.

The metabolic activation of 14C-labelled food carcinogens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to macromolecular bound species was studied in microsomal and hepatocellular incubations. Several data indicated that the covalent binding was dependent on P450 enzymes: It was dependent on NADPH, it was induced many times by the P450 IA1 and IA2 upregulators beta-naphthoflavone and polychlorinated biphenyls, and was inhibited by the P450 IA1 and IA2 inhibitor alpha-naphthoflavone. In both hepatocellular and microsomal incubations the three compounds bound with similar efficiency, with IQ being somewhat more potent compared to MeIQx and MeIQ. The binding appeared to follow saturation kinetics with Km values less than 20 microM. In incubations with hepatocytes the compounds bound to both cellular DNA and to bovine serum albumin in the medium. The fact that 13-26% of total adducts were formed with bovine serum albumin, indicates that reactive metabolites of the compounds may be transported and react at distant sites from their formation without any further activation.     

Covalent Binding of Food Carcinogens MeIQx,MeIQ and IQ to DNA and Protein in Microsomal Incubations and Isolated Rat Hepatocytes

Abstract: The metabolic activation of ¹⁴C-labelled food carcinogens 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4-dimethylimidazo[4, 5-f]quinoline (MeIQ) and 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ) to macromolecular bound species was studied in microsomal and hepatocellular incubations. Several data indicated that the covalent binding was dependent on P450 enzymes: It was dependent on NADPH, it was induced many times by the P450 IA1 and IA2 upregulators β-naphthoflavone and polychlorinated biphenyls, and was inhibited by the P450 IA1 and IA2 inhibitor α-naphthofiavone. In both hepatocellular and microsomal incubations the three compounds bound with similar efficiency, with IQ being somewhat more potent compared to MeIQx and MeIQ. The binding appeared to follow saturation kinetics with K_m values less than 20 μM. In incubations with hepatocytes the compounds bound to both cellular DNA and to bovine serum albumin in the medium. The fact that 13–26% of total adducts were formed with bovine serum albumin, indicates that reactive metabolites of the compounds may be transported and react at distant sites from their formation without any further activation.     

Plasma protein binding of phenol in dogs and rats as determined by equilibrium dialysis and ultrafiltration

Plasma-protein binding of phenol was examined in rats with and without ether anesthesia. Ether anesthesia decreased the percentage of phenol bound by rat plasma. The phenol binding capacities of dog and rat plasma were compared using equilibrium dialysis and ultrafiltration. For the wide range of phenol concentrations studied, dog plasma had a consistently higher binding capacity than rat plasma as demonstrated by both techniques. Higher binding was obtained by ultrafiltration than by equilibrium dialysis in both dog and rat plasma. Dog and rat serum albumins were responsible for 72–73% of the phenol binding in plasma, while bovine globulins either did not bind phenol or had only minimal binding capacities.     

The binding of colchicine and its derivatives to bovine and human serum albumin and human plasma

The protein binding of colchicine and its derivatives demecolcine and desacetylcolchiceine was studied by equilibrium dialysis at 22 degrees C and pH 7.38 in bovine and human serum albumin and human plasma. Colchicine and demecolcine (2 X 10(-4) -5 X 10(-4) mol/l) is not bound to proteins. The binding of desacetylcholchiceine was 60--80% in the range 10(-4)-5 X 10(-4) mol/l. The association constant for a single binding site was 8.03 X 10(3) 1/mol for human serum albumin and 13.20 X 10(3) 1/mol for human plasma. The binding profiles for desacetylcholchiceine were quite similar in human serum albumin (4% conc.) and human plasma (3.8% conc. of albumin fraction). We suggest that desacetylcolchiceine was likely to be bound predominantly to albumin. Salicylic acid in vitro, at clinical concentrations (1.8--14.5 X 10(-4) mol/l), significantly decreases the binding of desacetylcolchiceine to human serum albumin.     

Binding of some phenoxyalkanoic acids to bovine serum albumin in vitro.

The phenoxyalkanoix acids are bound to bovine serum albumin at a single site of high affinity, where the interaction is most importantly influenced by strong hydrophobic bonding, with ionic bonding being of lesser importance, and also at several sites of lower affinity. The hydrophobic binding site has almost equal affinity for the phenoxy groups of 2,4-dichlorophenoxyacetic acid, 4-(2,4-dichlorophenoxy)butyric acid and 2-(2,4,5-trichlorophenoxy)propionic acid and greater affinity for the phenoxy group of 2,4,5-trichlorophenoxyacetic acid. The properties of the high affinity binding site bear some resemblance to the properties of the amino acid sequence adjoining the tryptophan residue of human serum albumin, and evidence is presented for the presence of tryptophan at the high affinity binding site of bovine serum albumin.     

Glucuronide and Sulphate Binding to Subcellular Fractions of Rat Liver

Abstract The binding of glucuronides to various liver subfractions and to bovine serum albumin correlated with their molecular weight. Higher molecular weight glucuronides bound more readily. 4-Methylumbelliferyl-glucuronide had some of the characteristics of phenolphthaleinglucuronide. Simple phenolic glucuronides were much less retained by various hepatic fractions. Glucuronides were most effectively bound to mitochondrial subfractions. Sulphates of 4-nitrophenol and phenolphthalein were bound to the same degree to bovine serum albumin and hepatic cytosol despite their different molecular structures and weights.     

Binding of coumarin anticoagulants to human and bovine serum albumin. Circular dichroism studies

The interaction of acenocoumarin, coumachlor, phenprocoumon, and warfarin with human and bovine serum albumin was investigated by ultracentrifugation and circular dichroism measurements. Although all four drugs generate extrinsic Cotton effects when bound to human and bovine serum albumin, large differences in the signs and the intensities of the Cotton effects are observed. The differences in the induced Cotton effects suggest differential molecular binding mechanisms.