Hydrogen peroxide-mediated alteration of the heme prosthetic group of metmyoglobin to an iron chlorin product: evidence for a novel oxidative pathway.

Treatment of metmyoglobin with H2O2 is known to lead to the crosslinking of an active site tyrosine residue to the heme [Catalano, C. E., Y. S. Choe, and P. R. Ortiz de Montellano (1989) J. Biol. Chem. 264, 10534-10541]. We have found in this study that this reaction also leads to an altered heme product not covalently bound to the protein. This product was characterized by visible absorption, infrared absorption, and mass and NMR spectrometry as an iron chlorin product formed from the saturation of the double bond between carbon atoms at positions 17 and 18 of pyrrole ring D with concomitant addition of a hydroxyl group on the carbon atom at position 18 and lactonization of the propionic acid to the carbon atom at position 17. Studies with the use of (18)O-labeled H2O2, O2, and H2O clearly indicate that the source of the added oxygen on the heme is water. Evidently, water adds regiospecifically to a cationic site formed on a carbon atom at position 18 after oxidation of the ferric heme prosthetic group with peroxide. Prolonged incubation of the reaction mixture containing the iron hydroxychlorin product led to the formation of an iron dihydroxychlorin product, presumably from a slow addition of water to the initial iron hydroxychlorin. The iron chlorin products characterized in this study are distinct from the meso-oxyheme species, which is thought to be formed during peroxide-mediated degradation of metmyoglobin, cytochrome P450, ferric heme, and model ferric hemes, and give further insight into the mechanism of H2O2-induced heme alterations.

Inactivation of horseradish peroxidase by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine.

In the presence of H2O2, horseradish peroxidase (HRP) catalyzes the one-electron oxidation of a porphyrinogenic agent, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), leading to formation of an ethyl radical, inactivation of the enzyme, and formation of an altered heme product. The loss of heme during the inactivation of HRP was dependent on the duration of exposure to DDEP as well as the concentration of H2O2 and DDEP. The pseudo first order rate constant for the oxidation of DDEP by compound I of HRP at pH 7.4 was 0.07 min-1, and the maximal extent of heme loss was 35%. The altered heme product, which was isolated by reverse phase HPLC, was characterized by the use of mass and 1H NMR spectrometry as a substitution product of a C2H4OH moiety for a meso proton of the prosthetic heme [meso-(hydroxyethyl)heme]. The source of the oxygen in the C2H4OH moiety appeared not to be H2O2 or H2O as 18O was not incorporated in the heme adduct when H2(18)O2 or H2(18)O was used. The DDEP-mediated reaction did not form the expected delta-meso-ethylheme adduct analogous to the ethyl radical-mediated inactivation of HRP by ethylhydrazine (EH) [Ator et al. (1987) J. Biol. Chem. 262, 14954-14960]. However, we have found that meso-(hydroxyethyl)heme was formed in the EH-mediated reaction, albeit in apparently lower amounts than delta-meso-ethylheme. Perhaps the proximity of the heme to the ethyl radical may play a role in determining the nature of the heme products formed.

Structure of the novel heme adduct formed during the reaction of human hemoglobin with BrCCl3 in red cell lysates.

It was previously shown that the reductive debromination of BrCCl3 to trichloromethyl radical by human hemoglobin leads to formation of dissociable altered heme products, two of which are identical to those formed from myoglobin and one which is novel. In this study, we have elucidated the structure of this novel adduct with the use of mass spectrometry, as well as 1H and 13C NMR as a substitution product of a -C(Cl) = CCl2 moiety for a beta-hydrogen atom on the prosthetic heme's ring I vinyl group. From studies with the use of 13C-enriched BrCCl3, it was determined that the added carbon atoms were derived from 2 eq of BrCCl3. A mechanism that involves multiple reductive events and a radical cation heme intermediate is proposed. Consistent with this mechanism, cellular reductants were found to selectively enhance the amount of this novel dissociable heme adduct. These studies reveal fine differences between myoglobin and hemoglobin in the accessibility of reactive intermediates to the ring I vinyl group, as well as the potential importance of cellular reductants on the course of heme alteration.

Immunochemical detection of liver protein adducts of the nonsteroidal antiinflammatory drug diclofenac.

Serious idiosyncratic hepatic injury has been associated with the use of many nonsteroidal antiinflammatory drugs, including the widely prescribed agent diclofenac. In order to investigate the possibility that covalent protein adducts of reactive metabolites of diclofenac might be responsible for the hepatotoxicity produced by this drug, we have developed a polyclonal antibody that can recognize such adducts in tissues. Immunoblotting revealed that protein adducts of reactive metabolites of diclofenac of 50, 70, 110, and 140 kDa were formed in the livers of mice treated with diclofenac. In the future, it will be determined whether these adducts can cause hepatotoxicity by either a hypersensitivity or metabolic mechanism. Similar approaches may be used to study the protein adducts and mechanisms of hepatotoxicity of other nonsteroidal antiinflammatory drugs.

The use of stable isotopes to identify reactive metabolites and target macromolecules associated with toxicities of halogenated hydrocarbon compounds.

1. Halogenated compounds, such as the inhalation anaesthetics, halothane and enflurane, and the chemicals chloroform, carbon tetrachloride, and bromotrichloromethane can cause hepatotoxicity, nephrotoxicity, and inactivation of cytochromes P-450. Each of these toxicities is mediated by reactive metabolites. 2. Stable isotopes of hydrogen, carbon, chlorine and oxygen have been used in conjunction with mass spectrometry and n.m.r. spectrometry to identify the structures of these metabolites, to elucidate the mechanisms of their formation, and to characterize the structures of their macromolecular adducts. 3. In a number of cases, oxidative pathways of metabolism to toxic metabolites have been defined by kinetic deuterium isotope effects. 4. Recently, we have found that the trichloromethyl radical metabolite of bromotrichloromethane can activate myoglobin by causing the covalent cross-linking of haem to protein. The structure of a haem-myoglobin adduct has been defined by the use of stable isotope studies.

Characterization by NMR of the heme-myoglobin adduct formed during the reductive metabolism of BrCCl3. Covalent bonding of the proximal histidine to the ring I vinyl group.

The reductive debromination of BrCCl3 by ferrous deoxymyoglobin leads to the covalent bonding of the prosthetic heme to the protein. We have previously shown, by the use of peptide mapping and mass spectrometry, that histidine residue 93 is covalently bound to the heme moiety. In the present study the structure of the heme adduct was more completely determined by 1H and 13C NMR techniques. We have found that the ring I vinyl group of the prosthetic heme was altered by the addition of a histidine imidazole nitrogen to the alpha-carbon and a CCl2 moiety to the beta-carbon. The electronic absorption spectra of the oxidized and reduced states of the altered heme-protein indicated that the heme-iron exists in a bis-histidine-ligated form. Analysis of the crystal structure of native myoglobin suggested that for the altered heme-protein, histidine residues 97 and 64 are ligated to the heme-iron and that residue 97 has replaced the native proximal histidine residue 93. These movements, in effect a "histidine shuffle" at the active site, may be responsible for the enhanced reducing activity of the altered protein.

Oxidative cyclization, 1,4-benzothiazine formation and dimerization of 2-bromo-3-(glutathion-S-yl)hydroquinone.

Several lines of evidence suggest that the renal-specific toxicity of quinol-linked GSH conjugates is probably a result of their metabolism by gamma-glutamyl transpeptidase and selective accumulation by proximal tubular cells. Transport of the resultant quinol-cysteine and/or cystein-S-ylglycine conjugate followed by oxidation to the quinone may be important steps in the mechanism of toxicity of these compounds. Factors modulating the intracellular and/or intralumenal concentration of the cystein-S-yl and cystein-S-ylglycine conjugate will, therefore, be important determinants of toxicity. We have now studied the gamma-glutamyl transpeptidase-mediated metabolism of 2-bromo-3-(glutathion-S-yl)hydroquinone. The product of this reaction, 2-bromo-3-(cystein-S-ylglycyl)hydroquinone, undergoes an intramolecular cyclization to yield a 1,4-benzothiazine derivative that retains the glycine residue. A similar cyclization reaction occurs with 2-bromo-3-(cystein-S-yl)hydroquinone, which is unstable in aqueous solutions and undergoes a pH-dependent rearrangement that requires initial oxidation to the quinone. UV spectroscopy revealed that, at neutral pH, further reaction results in the formation of a chromophore, consistent with 1,4-benzothiazine formation. This product arises via cyclization of the cysteine residue via an intramolecular 1,4 Michael addition. Further reaction results in the precipitation of a pigment that exhibits properties of a pH indicator. The pigment undergoes a marked pH-dependent bathochromic shift (approximately 100 nm); it is red in alkali (lambda max, 480 nm) and violet in acid (lambda max, 578 nm). These properties are similar to those of the trichochrome polymers that are formed during melanin biosynthesis from S-(3,4-dihydroxyphenylalanine)-L-cysteine. Because the intramolecular cyclization reactions remove the reactive quinone moiety from the molecules, they may be regarded as detoxication reactions. 1,4-Benzothiazine formation represents a novel pathway that diverges from the usual route of mercapturic acid synthesis and may represent previously unrecognized and important products of quinone metabolism in vivo.

Differences in the localization and extent of the renal proximal tubular necrosis caused by mercapturic acid and glutathione conjugates of 1,4-naphthoquinone and menadione.

We have previously demonstrated that administration of various benzoquinol-glutathione (GSH) conjugates to rats causes renal proximal tubular necrosis and the initial lesion appears to lie within that portion of the S3 segment within the outer stripe of the outer medulla (OSOM). The toxicity may be a consequence of oxidation of the quinol conjugate to the quinone followed by covalent binding to tissue macromolecules. We have therefore synthesized the GSH and N-acetylcysteine conjugates of 2-methyl-1,4-naphthoquinone (menadione) and 1,4-naphthoquinone. The resulting conjugates have certain similarities to the benzoquinol-GSH conjugates, but the main difference is that reaction with the thiol yields a conjugate which remains in the quinone form. 2-Methyl-3-(N-acetylcystein-S-yl)-1,4-naphthoquinone caused a dose-dependent (50-200 mumol/kg) necrosis of the proximal tubular epithelium. The lesion involved the terminal portion of the S2 segment and the S3 segment within the medullary ray. At the lower doses, that portion of the S3 segment in the outer stripe of the outer medulla displayed no evidence of necrosis. In contrast, 2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone (200 mumol/kg) caused no apparent histological alterations to the kidney. 2-(Glutathion-S-yl)-1,4-naphthoquinone and 2,3-(diglutathion-S-yl)-1,4-naphthoquinone (200 mumol/kg) were relatively weak proximal tubular toxicants and the lesion involved the S3 segment at the junction of the medullary ray and the OSOM. A possible reason(s) for the striking difference in the toxicity of the N-acetylcysteine conjugate of menadione, as opposed to the lack of toxicity of the GSH conjugate of menadione, is discussed. The basis for the localization of the lesion caused by 2-methyl-3-(N-acetylcystein-S-yl)-1,4-naphthoquinone requires further study.

Sequential oxidation and glutathione addition to 1,4-benzoquinone: correlation of toxicity with increased glutathione substitution.

The chemical reaction of 1,4-benzoquinone with glutathione results in the formation of adducts that exhibit increasing degrees of glutathione substitution. Purification of these adducts and analysis by 1H and 13C nuclear magnetic resonance spectroscopy revealed the products of the reaction to be 2-(glutathion-S-yl)hydroquinone; 2,3-(diglutathion-S-yl)hydroquinone; 2,5-(diglutathion-S-yl)hydroquinone; 2,6(diglutathion-S-yl)hydroquinone; 2,3,5-(triglutathion-S-yl)hydroquinone; and 2,3,5,6-(tetraglutatathion-S-yl)hydroquinone. The initial conjugation of 1,4-benzoquinone with glutathione did not significantly affect the oxidation potential of the compound. However, subsequent oxidation and glutathione addition resulted in the formation of conjugates that, dependent upon the position of addition, become increasingly more difficult to oxidize. Increased glutathione substitutions, which resulted in an increase in oxidation potentials, paradoxically resulted in enhanced nephrotoxicity. The triglutathion-S-yl conjugate was the most potent nephrotoxicant; the diglutathion-S-yl conjugates exhibited similar degrees of nephrotoxicity; the mono- and tetraglutathion-S-yl conjugates were not toxic. Thus, with the exception of the fully substituted isomer, the severity of renal necrosis correlated with the extent of glutathione substitution. The lack of toxicity of the fully substituted isomer is probably a consequence of its inability to alkylate tissue components. Thus, the conjugation of glutathione with quinones does not necessarily result in detoxification, even when the resulting conjugates are more stable to oxidation. The inhibition of gamma-glutamyl transpeptidase by AT-125 protected against 2,3,5-(triglutathion-S-yl)hydroquinone-mediated nephrotoxicity. It is suggested that other extra-renal sites expressing relatively high levels of gamma-glutamyl transpeptidase might therefore also be susceptible to hydroquinone-linked glutathione conjugate toxicity. This pathway might also contribute to the carcinogenicity and mutagenicity of certain quinones.

2-Bromo-(diglutathion-S-yl)hydroquinone nephrotoxicity: physiological, biochemical, and electrochemical determinants.

2-Bromo-(diglutathion-S-yl)hydroquinone [2-Br-(diGSyl)HQ] causes severe necrosis of the proximal renal tubules in the rat, elevations in blood urea nitrogen (BUN) and increased urinary excretion of protein, glucose, and lactate dehydrogenase. In contrast, 2-Br-3-(GSyl)HQ, 2-Br-5-(GSyl)HQ, and 2-Br-6-(GSyl)HQ caused differentially less toxicity than the diglutathionyl conjugate. None of these conjugates had any apparent effect on liver pathology and serum glutamate-pyruvate transaminase remained within the normal range. Pretreatment of rats with probenecid, an organic anion transport inhibitor, offered only slight protection against 2-Br-(diGSyl)HQ-mediated elevations in BUN, proteinuria, or glucosuria. In contrast, quinine, an organic cation transport inhibitor, potentiated the nephrotoxicity of 2-Br-(di-GSyl)HQ. Thus, in contrast to other nephrotoxic sulfur conjugates, probenecid-sensitive organic ion transport systems do not contribute to the kidney-specific toxicity of 2-Br-(diGSyl)HQ. However, inhibition of renal gamma-glutamyl transpeptidase by AT-125 completely protected rats from the nephrotoxic effects of 2-Br-(diGSyl)HQ. Aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, caused a 20-25% decrease in 2-Br-(diGSyl)HQ-mediated elevations in BUN and urinary excretion parameters. The isomeric 35S conjugates covalently bound to rat kidney 10,000 x g homogenate in the order 2-Br-6-(GSyl)HQ greater than 2-Br-5-(GSyl)HQ greater than 2-Br-3-(GSyl)HQ greater than 2-Br-(diGSyl)HQ. AT-125 (0.4 mM) decreased covalent binding by 25%, 17%, 33%, and 28%, respectively. Aminooxyacetic acid (0.1 mM) inhibited covalent binding by 26%, 10%, 17%, and 17% respectively. Ascorbic acid (1.0 mM) inhibited covalent binding by 63%, 87%, 62%, and 28%, respectively, and this inhibition correlated, inversely, with the redox potential of the conjugates. Thus, the covalent binding is mediated preferentially by oxidation of the quinol moiety, although the formation of reactive thiols cannot be excluded. In addition, the initial conjugation of 2-BrHQ with GSH does not result in the formation of a less redox-active species. However, the subsequent addition of a second molecule of GSH results in the formation of a more redox-stable compound, which, paradoxically, enhances toxicity. The metabolism of 2-Br-(diGSyl)HQ by renal proximal tubular gamma-glutamyl transpeptidase and trans-membrane transport of the cysteine conjugate(s) followed by oxidation of the quinol moiety is probably responsible for the target organ toxicity of this compound.

Synthesis and nephrotoxicity of 6-bromo-2,5-dihydroxy-thiophenol.

The formation of potentially reactive thiols has been postulated to play a role in the nephrotoxicity caused by a number of glutathione and/or cysteine conjugates. However, the inherent reactivity of such compounds has precluded both their identification in biological systems and a determination of their actual toxicity. To this end we have synthesized 6-bromo-2,5-dihydroxy-thiophenol as a putative metabolite of nephrotoxic 2-bromohydroquinone-glutathione conjugates. The compound was prepared by the addition of sodium thiosulfate to 2-bromo-1,4-benzoquinone followed by reduction of the S-arylthiosulfate to the thiophenol. 2,5-Dihydroxy-thiophenol was similarly prepared. Structural identification was confirmed by mass spectroscopy and nuclear magnetic resonance spectroscopy. Administration of 6-bromo-2,5-dihydroxy-thiophenol to rats (0.35 mmol/kg; intraperitoneally) caused an increase in blood urea nitrogen and histological alterations similar to those observed after 2-bromo-(diglutathion-S-yl)hydroquinone administration. 2,5-Dihydroxy-thiophenol was also nephrotoxic but at a dose of 0.6 mmol/kg. In contrast, no effects on liver pathology were observed after administration of either 6-bromo-2,5-dihydroxy-thiophenol or 2,5-dihydroxy-thiophenol and serum glutamate pyruvate transaminase levels were normal. Neither 2-, 3-, nor 4-bromothiophenol had any effect on blood urea nitrogen at doses between 0.2 and 0.8 mmol/kg (intraperitoneally) and no apparent alterations were seen in kidney slices prepared from bromothiophenol-treated rats. These findings suggest that the quinone function of 6-bromo-2,5-dihydroxy-thiophenol is necessary for the expression of toxicity. In this respect, the lower activity of NAD(P)H quinone oxidoreductase (EC 1.6.99.2) in renal cortex may be of toxicological significance.

Metasternal gland secretion of the locust tree borer, Megacyllene robiniae (Coleoptera: Cerambycidae).

1. The volatile components of metasternal gland extracts of male and female Megacyllene robiniae have been analyzed by gas chromatography-mass spectroscopy. 2. The major component was identified as 2-(1,3-hexadien-1-yl)-5-methyltetrahydrofuran, a new natural product. 3. 1-Phenylethanol is present only in male extracts. 4. Acetates of hexadecanol and octadecanol are also present.

Mutagenic perylenequinone metabolites of Alternaria alternata: altertoxins I, II, and III.

The mold genus Alternaria is a widely distributed plant pathogen. Some of these species, e.g., A. alternata, are common decay organisms of fruits and vegetables. Two novel perylene oxide metabolites, altertoxins II and III, have been identified in extracts of A. alternata isolates that exhibit mutagenic responses in the Ames Salmonella typhimurium assay. These identifications were based on mass, optical rotational, and 1H- and 13C-nmr spectral studies. Previous reports of related perylene dione mycotoxins have been clarified.

Alkaloids from dendrobatid frogs: structures of two omega-hydroxy congeners of 3-butyl-5-propylindolizidine and occurrence of 2,5-disubstituted pyrrolidines and a 2,6-disubstituted piperidine.

Forty alkaloids were detected and characterized from skin extracts of high- and low-elevation populations of the poison frog Dendrobates histrionicus from northwestern Colombia. Combined gc/ms with NH3 or ND3 in a chemical ionization mode detected protonated parent ions and determined the number of exchangeable NH and OH hydrogens. Six previously unknown dendrobatid alkaloids were characterized. Two were 2,5-disubstituted pyrrolidines, which included pyrrolidine 197B, a trans-2-butyl-5-pentylpyrrolidine, while a third was a 2,6-dipentylpiperidine. Indolizidines 239AB and 239CD had the same relative configuration as the parent alkaloid 223AB [(5E,9E)3-butyl-5-propylindolizidine] and contained, respectively, a omega-hydroxy group in the propyl or butyl side chain. The profiles of alkaloids in the new northern populations of D. histrionicus are typical of the species in containing a set of about eight histrionicotoxins, in marked contrast to a related species, Dendrobates lehmanni, which does not contain histrionicotoxins.

Glutathione conjugates of 2-bromohydroquinone are nephrotoxic.

Incubation of either o-bromophenol or 2-bromohydroquinone with rat liver microsomes and 0.25 mM 35S-glutathione (GSH) gave rise to several isomeric 35S-GSH conjugates. A mixture of these isomeric GSH conjugates was prepared chemically and two were purified by HPLC; 1H-NMR spectroscopy revealed that one was 2-bromo-3-(glutathion-S-yl)hydroquinone and the other was a disubstituted GSH conjugate which could be either 2-bromo-3,5-(diglutathion-S-yl)hydroquinone or 2-bromo-3,6-(diglutathion-S-yl)hydroquinone. Injection of the disubstituted GSH conjugate intravenously to rats caused substantial elevations in blood urea nitrogen levels. Treatment of rats with AT-125 (Acivicin; NSC 163501; 10 mg/kg ip) caused a substantial inhibition of kidney gamma-glutamyl transpeptidase activity and decreased 2-bromohydroquinone-mediated elevations in blood urea nitrogen. These findings are consistent with the view that the kidney necrosis observed after administration of either bromobenzene (1), o-bromophenol (2), or 2-bromohydroquinone (3) might be due in part to 2-bromohydroquinone GSH conjugates formed in the liver and subsequently transported to the kidney and converted to ultimate nephrotoxic metabolite(s).

Two-dimensional J-resolved nuclear magnetic resonance spectral study of two bromobenzene glutathione conjugates.

The application of two-dimensional J-resolved nuclear magnetic resonance spectroscopy to determine the structure of two bile metabolites isolated from rats injected interperitoneally with bromobenzene is described. The structures of the two molecules are obtained unambiguously from the proton-proton spin coupling constants. This paper discusses the fundamentals of the technique and demonstrates the resolution of small long-range coupling constants.

Formation of nontoxic reactive metabolites of p-bromophenol. Identification of a new glutathione conjugate.

A microsomal metabolite of p-bromophenol was isolated and identified as 6-(glutathion-S-yl)-4-bromocatechol. p-Bromophenol is metabolized in rat liver microsomes in part to 4-bromocatechol. The catechol undergoes autooxidation to the corresponding quinone or semiquinone, which can either covalently bind to microsomal protein or, in the presence of glutathione, form a glutathione conjugate. Superoxide dismutase inhibited these reactions by preventing the superoxide anion-mediated oxidation of 4-bromocatechol. Thus, in the presence of glutathione, superoxide dismutase caused a decrease in conjugate formation with a corresponding increase in 4-bromocatechol levels. Conditions which increased the in vitro covalent binding of p-bromophenol (namely, phenobarbital treatment and the absence of glutathione) did not cause toxicity in vivo. Thus, chemically reactive metabolite(s) of p-bromophenol do not play a role in bromobenzene-mediated hepatotoxicity.

Reductive-oxygenation mechanism of metabolism of carbon tetrachloride to phosgene by cytochrome P-450.

The mechanism of metabolism of carbon tetrachloride (CCl4) to phosgene (COCl2) in rat liver microsomes was investigated. When the oxygen dependency of the reaction was studied, it was found that the rate of the reaction increased as the oxygen concentration in the reaction atmosphere was decreased from 100% to 5%. Decreasing the oxygen concentration below 5% caused a decrease in the rate of the reaction. The reaction was not inhibited by superoxide dismutase or catalase nor was it supported by cumene hydroperoxide. A reconstituted form of cytochrome P-450 from phenobarbital-pretreated rats metabolized CCl4 to COCl2. These results are consistent with a mechanism we call reductive oxygenation. The first step of the reaction is the cytochrome P-450-dependent reductive dechlorination of CCl4 to trichloromethyl radical (CCl3.). This intermediate is trapped by oxygen to form trichloromethylperoxyl radical (CCl3OO.), which decomposes to COCl2 and possibly an electrophilic form of chlorine.