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PubMed2002

Comparative modeling of the latent form of a plant catechol oxidase using a molluskan hemocyanin structure.

The structure of the precursor form of catechol oxidase from sweet potatoes (Ipomoea batatas) has been modeled on the basis of the 3D structural data of mature catechol oxidase [Nat. Struct. Biol. 5 (1998) 1084] and of hemocyanin from giant octopus (Octopus dofleini) [J. Mol. Biol. 278 (1998) 855]. A C-terminal extension peptide is found in the cDNA sequence but not in the purified, mature form of catechol oxidase. Superimposition of the 3D structures of the native hemocyanin and catechol oxidase reveals a close relationship except for an additional C-terminal domain only found in the hemocyanin structure. As sequence alignment shows good homology this domain of the hemocyanin structure was used as a template to model the 3D structure of the C-terminal extension peptide of catechol oxidase. As hemocyanins show no or only weak catecholase activity due to this domain this indicates an inhibitory function of this extension peptide. Beside this possible shielding function for the precursor form, evidence for a function in copper-uptake also increases due to the location of three histidine residues in the model.

Amino Acid Sequence↗
PubMed1999

Effects of low dose catechol on glandular stomach carcinogenesis in BALB/c mice initiated with N-methyl-N-nitrosourea.

The effects of low dose catechol administration in the diet on stomach carcinogenesis in mice after initiation with N-methyl-N-nitrosourea (MNU) in the drinking water were investigated. Male, 6-week-old, BALB/c mice were given MNU in the drinking water intermittently for 1-week periods at 1-week intervals for a total of 3 weeks at a concentration of 120 ppm (groups 1 and 3). Groups 2 and 4 served as non-initiated controls. From week 7, groups 1 and 3 were divided into four subgroups and the mice were fed on a diet containing 4 ppm (groups la and 3a), 20 ppm (groups 1b and 3b), 100 ppm (groups 1c and 3c), 500 ppm (groups 1d and 3d) or 0 ppm (groups 2 and 4) catechol for 44 weeks. At week 50, appreciably enhanced development of pepsinogen 1 altered pyloric glands (PAPG) was noted in groups 1c and 1d. The incidences of adenomatous hyperplasia and carcinomas were not affected in any of the catechol-treated groups as compared with corresponding controls on a basal diet. Thus, the administration of catechol in the diet at low doses enhanced only preneoplastic lesion development and not neoplastic lesion development. From these results, we conclude that the biological significance of the catechol promoting effect at probable human exposure levels on gastric cancer is probably limited, while the PAPG may be a sensitive endpoint lesion for mouse glandular stomach carcinogenesis.

Adenocarcinoma↗
PubMed1999

Radiochemical high-performance liquid chromatographic assay for the determination of catechol O-methyltransferase activity towards various substrates.

A new chromatographic catechol O-methyltransferase (COMT) assay based on S-adenosyl-L-[methyl-14C]methionine and on-line radioactivity detection was developed. With minor modifications in the mobile phase composition the methylation velocities for 30 structurally diverse compounds including simple catechols, neurotransmitters, catecholestrogens and catecholic drugs could be measured using human and rat recombinant soluble COMT. The enzymes showed very similar substrate selectivities. The radiochemical method was validated using 3,4-dihydroxybenzoic acid as a model substrate and it was shown that accurate and reproducible methylation velocity values could be achieved for both of the catecholic hydroxyls. The method proved to be suited for determining the enzyme kinetic parameters and can probably be further used for gathering enzyme kinetic data on differentially substituted catechols in order to construct proper structure-activity relationships for COMT.

Animals↗
PubMed1998

Synergistic induction of DNA strand breakage by catechol-estrogen and nitric oxide: implications for hormonal carcinogenesis.

Estrogen is a known risk factor for human breast cancer, although the mechanism by which estrogens induce cancer remains unestablished. We have demonstrated that DNA strand breakage is induced synergistically when pBR322 plasmid DNA is incubated in the presence of both a nitric oxide (NO)-releasing compound (diethylamine NONOate, etc.) and a catechol-estrogen (2- or 4-hydroxyestradiol or -hydroxyestrone). Either the NO-releasing compound or the catechol-estrogen alone induced much fewer strand breaks. Estradiol, estrone, O-methylated catechol-estrogens, and diethylstilbestrol did not exert such DNA damaging effects. Strand breakage induced by NO plus 2- or 4-hydroxyestradiol was inhibited by carboxy-PTIO (an NO-trapping agent) and, to a lesser extent, by superoxide dismutase. Antioxidants (e.g., N-acetylcysteine, ascorbate), but not HO. scavengers, exhibited inhibitory effects. A possible mechanism for this strand breakage would be: (1) NO mediates conversion of catechol-estrogens to quinones, (2) the quinone/hydroquinone redox system produces O2.-, and (3) O2.- reacts with NO to form peroxynitrite, which causes DNA strand breaks. Our results imply that interaction of catechol-estrogens and NO, both known to be formed in human breast and uterus, leads to production of a potent oxidant(s), which could cause damage in cells and DNA, thus playing an important role in hormonal carcinogenesis.

Antioxidants↗
PubMed2003

Kinetics of anaerobic biodegradation of resorcinol catechol and hydroquinone in upflow fixed film-fixed bed reactors.

Biodegradation of resorcinol, catechol and hydroquinone under anaerobic conditions was studied in identical upflow fixed film-fixed bed reactors. Kinetic constants; V(max) (maximum substrate utilization rate) and K(s) (Monod's half saturation constant) were determined for the three compounds using Lineweaver-Burk plot. V(max) for resorcinol was highest, followed by catechol and then by hydroquinone. When both resorcinol and catechol were fed to the resorcinol-acclimated reactor, resorcinol degradation was inhibited by catechol. The inhibition was of the uncompetitive type and V(max) for resorcinol was reduced by catechol.

Antioxidants↗
PubMed2003

Novel iron(III) complexes of tripodal and linear tetradentate bis(phenolate) ligands: close relevance to intradiol-cleaving catechol dioxygenases.

Four new iron(III) complexes of the bis(phenolate) ligands N,N-dimethyl-N',N'-bis(2-hydroxy-3,5-dimethylbenzyl)ethylenediamine [H2(L1)], N,N-dimethyl-N',N'-bis(2-hydroxy-4-nitrobenzyl)ethylenediamine [H2(L2)], N,N'-dimethyl-N,N'-bis(2-hydroxy-3,5-dimethylbenzyl)ethylenediamine [H2(L3)], and N,N'-dimethyl-N,N'-bis(2-hydroxy-4-nitrobenzyl)ethylenediamine [H2(L4)] have been isolated and studied as structural and functional models for the intradiol-cleaving catechol 1,2-dioxygenases (CTD). The complexes [Fe(L1)Cl] (1), [Fe(L2)(H2O)Cl] (2), [Fe(L3)Cl] (3), and [Fe(L4)(H2O)Cl] (4) have been characterized using absorption spectral and electrochemical techniques. The single-crystal X-ray structures of the ligand H2(L1) and the complexes 1 and 2 have been successfully determined. The tripodal ligand H2(L1) containing a N2O2 donor set represents the metal-binding region of the iron proteins. Complex 1 contains an FeN2O2Cl chromophore with a novel trigonal bipyramidal coordination geometry. While two phenolate oxygens and an amine nitrogen constitute the trigonal plane, the other amine nitrogen and chloride ion are located in the axial positions. In contrast, 2 exhibits a rhombically distorted octahedral coordination geometry for the FeN2O3Cl chromophore. Two phenolate oxygen atoms, an amine nitrogen atom, and a water molecule are located on the corners of a square plane with the axial positions being occupied by the other nitrogen atom and chloride ion. The interaction of the complexes with a few monodentate bases and phenolates and differently substituted catechols have been investigated using absorption spectral and electrochemical methods. The effect of substituents on the phenolate rings on the electronic spectral features and FeIII/FeII redox potentials of the complexes are discussed. The interaction of the complexes with catecholate anions reveals changes in the phenolate to iron(III) charge-transfer band and also the appearance of a low-energy catecholate to iron(III) charge-transfer band similar to catechol dioxygenase-substrate complexes. The redox behavior of the 1:1 adducts of the complexes with 3,5-di-tert-butylcatechol (H2DBC) has been also studied. The reactivities of the present complexes with H2DBC have been studied and illustrated. Interestingly, only 2 and 4 catalyze the intradiol-cleavage of H2DBC, the rate of oxygenation being much faster for 4. Also 2, but not 4, yields an extradiol cleavage product. The reactivity of the complexes could be illustrated not on the basis of the Lewis acidity of the complexes alone but by assuming that the product release is the rate-determining phase of the catalytic reaction.

Catechol 1,2-Dioxygenase↗
PubMed2004

Iron(III) complexes of sterically hindered tetradentate monophenolate ligands as functional models for catechol 1,2-dioxygenases: the role of ligand stereoelectronic properties.

The iron(III) complexes of the monophenolate ligands 2-(bis(pyrid-2-ylmethyl)aminomethyl)-4-nitrophenol [H(L1)], N,N-dimethyl-N'-(pyrid-2-ylmethyl)-N'-(2-hydroxy-4-nitrobenzyl)ethylenediamine [H(L2)], N,N-dimethyl-N'-(6-methyl-pyrid-2-ylmethyl)-N'-(2-hydroxy-4-nitrobenzyl)ethylenediamine [H(L3)], and N,N-dimethyl-N'-(1-methylimidazole-2-ylmethyl)-N'-(2-hydroxy-4-nitrobenzyl)ethylenediamine [H(L4)] have been obtained and studied as structural and functional models for the intradiol-cleaving catechol dioxygenase enzymes. The complexes [Fe(L1)Cl(2)].CH(3)CN (1), [Fe(L2)Cl(2)] (2), [Fe(L3)Cl(2)] (3), and [Fe(L4)Cl(2)] (4) have been characterized using absorption spectral and electrochemical methods. The single crystal X-ray crystal structures of 1 and 2 have been successfully determined. Both the complexes possess a rhombically distorted octahedral coordination geometry for the FeN(3)OCl(2) chromophore. In 2, the phenolate oxygen, the pyridine nitrogen, an amine nitrogen, and a chloride ion are located on the corners of a square plane with the nitrogen atom of a -NMe(2) group and the other chloride ion occupying the axial positions. In 1, also the equatorial plane is constituted by the phenolate oxygen, the pyridine nitrogen, an amine nitrogen atom, and a chloride ion; however, the axial positions are occupied by the second pyridine nitrogen and the second chloride ion. Interestingly, the Fe-O-C angle of 136.1 degrees observed for 2 is higher than that (128.5 degrees ) in 1; however, the Fe-O(phenolate) bond distances in both the complexes are the same (1.929 A). This illustrates the importance of the nearby sterically demanding coordinated -NMe(2) group and implies similar stereochemical constraints from the other ligated amino acid moieties in the 3,4-PCD enzymes, the enzyme activity of which is traced to the difference in the equatorial and axial Fe-O(tyrosinate) bonds (Fe-O-C, 133 degrees, 148 degrees ). The nature of heterocyclic rings of the ligands and the methyl substituents on them regulates the electronic spectral features, Fe(III)/Fe(II) redox potentials, and catechol cleavage activity of the complexes. Upon interacting the complexes with catecholate anions, two catecholate to iron(III) charge transfer bands appear, and the low energy band is similar to that of catechol dioxygenase-substrate complex. Complexes 1 and 3 fail to catalyze the oxidative intradiol cleavage of 3,5-di-tert-butylcatechol (H(2)DBC). However, interestingly, the replacement of pyridine pendant in 1 by the -NMe(2) group to obtain 2 restores the dioxygenase activity, which is consistent with its higher Fe-O-C bond angle. Remarkably, the more basic N-methylimidazole ring in 4 facilitates the rate-determining product releasing phase of the catalytic reaction, leading to enhancement in reaction rate and efficient conversion (77.1%) of the substrate to intradiol cleavage products as well. All these observations provide support to the novel substrate activation mechanism proposed for the intradiol-cleavage pathway.

Catalysis↗
PubMed2001

Synthesis and reactivity of the catechol metabolites from the equine estrogen, 8,9-dehydroestrone.

The risk factors for women developing breast and endometrial cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy in particular has been correlated with an increased cancer risk. Previously, we showed that the equine estrogens equilin and equilenin, which are major components of the widely prescribed estrogen replacement formulation Premarin, are metabolized to highly cytotoxic quinoids which caused oxidative stress and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. Chem. Res. Toxicol. 1998, 11, 1113-1127]. In this study, we have synthesized 8,9-dehydroestrone (a third equine estrogen component of Premarin) and its potential catechol metabolites, 4-hydroxy-8,9-dehydroestrone and 2-hydroxy-8,9-dehydroestrone. Both 2-hydroxy-8,9-dehydroestrone and 4-hydroxy-8,9-dehydroestrone were oxidized by tyrosinase or rat liver microsomes to o-quinones which reacted with GSH to give one mono-GSH conjugate and two di-GSH conjugates. Like endogenous estrogens, 8,9-dehydroestrone was primarily converted by rat liver microsomes to the 2-hydroxylated rather than the 4-hydroxylated o-quinone GSH conjugates; the ratio of 2-hydroxy-8,9-dehydroestrone versus 4-hydroxy-8,9-dehydroestrone was 6:1. Also in contrast to experiments with equilin, 4-hydroxyequilenin was not observed in microsomal incubations with 8,9-dehydroestrone or its catechols. The behavior of 2-hydroxy-8,9-dehydroestrone was found to be more complex than 4-hydroxy-8,9-dehydroestrone as GSH conjugates resulting from 2-hydroxy-8,9-dehydroestrone were detected even without oxidative enzyme catalysis. Under physiological conditions, 2-hydroxy-8,9-dehydroestrone isomerized to 2-hydroxyequilenin to form the very stable 2-hydroxyequilenin catechol; however, 4-hydroxy-8,9-dehydroestrone was found to be stable under similar conditions. Finally, preliminary studies conducted with the human breast tumor S-30 cell lines demonstrated that the catechol metabolites of 8,9-dehydroestrone were much less toxic than 4-hydroxyequilenin (20-40-fold). These results suggest that the catechol metabolites of 8,9-dehydroestrone may have the ability to cause cytotoxicity in vivo primarily through formation of o-quinones; however, most of the adverse effects of Premarin estrogens are likely due to formation of 4-hydroxyequilenin o-quinone from equilin and equilenin.

Animals↗
PubMed2006

Synthetic models of the active site of catechol oxidase: mechanistic studies.

The ability of copper proteins to process dioxygen at ambient conditions has inspired numerous research groups to study their structural, spectroscopic and catalytic properties. Catechol oxidase is a type-3 copper enzyme usually encountered in plant tissues and in some insects and crustaceans. It catalyzes the conversion of a large number of catechols into the respective o-benzoquinones, which subsequently auto-polymerize, resulting in the formation of melanin, a dark pigment thought to protect a damaged tissue from pathogens. After the report of the X-ray crystal structure of catechol oxidase a few years earlier, a large number of publications devoted to the biomimetic modeling of its active site appeared in the literature. This critical review (citing 114 references) extensively discusses the synthetic models of this enzyme, with a particular emphasis on the different approaches used in the literature to study the mechanism of the catalytic oxidation of the substrate (catechol) by these compounds. These are the studies on the substrate binding to the model complexes, the structure-activity relationship, the kinetic studies of the catalytic oxidation of the substrate and finally the substrate interaction with (per)oxo-dicopper adducts. The general overview of the recognized types of copper proteins and the detailed description of the crystal structure of catechol oxidase, as well as the proposed mechanisms of the enzymatic cycle are also presented.

Binding Sites↗
PubMed1968

Catechol oxygenase induction in Pseudomonas aeruginosa.

1. The transfer from benzenesulphonate to benzoate as a growth substrate for Pseudomonas aeruginosa strain A resulted in a change in the enzymic route by which catechol was degraded; at intermediate stages it was possible to obtain cells containing the enzymes of both the ;ortho' and ;meta' metabolic pathways. 2. A similar result was effected by the reverse transfer, benzoate to benzenesulphonate. 3. Catechol itself always elicited a catechol 2,3-oxygenase in uninduced cells, but the product of this reaction, 2-hydroxymuconic semialdehyde, and biochemically related compounds such as 4-hydroxy-2-oxovalerate, unexpectedly induced a catechol 1,2-oxygenase. 4. Both types of catechol oxygenase are strongly repressed by the metabolic end products of both the ;ortho' and ;meta' pathways, but there was no inhibition of enzymic activity by these end products.

Benzoates↗
PubMed1999

Catechol dioxygenases.

Catechol dioxygenases are key enzymes in the metabolism of aromatic rings by soil bacteria. Catechol dioxygenases have been found that participate in the metabolism of halogenated aromatic compounds and, in doing so, play a key role in bioremediation of halogenated pollutants. The catechol dioxygenases can be divided into two major groups: those that cleave the aromatic ring between the vicinal diols (the intradiol enzymes) and those that cleave the ring to one side of the vicinal diols (the extradiol enzymes). Whereas both types of catechol dioxygenase contain an active-site iron that is required absolutely for enzymic activity, the intradiol enzymes contain Fe(III), while the extradiol enzymes contain Fe(II). The nature of the protein ligands determines this specificity. The differences in oxidation state of the active-site iron appear to result in mechanistic differences that lead to the differing regioselectivity of the two groups of catechol dioxygenase. Mechanistic proposals based on available evidence suggest a substrate-activation mechanism for the intradiol enzymes and an oxygen-activation mechanism for the extradiol enzymes.

Bacteria↗
PubMed2001

The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase by catecholic substrates.

2,3-Dihydroxybiphenyl 1,2-dioxygenase (EC ), the extradiol dioxygenase of the biphenyl biodegradation pathway, is subject to inactivation during the steady-state cleavage of catechols. Detailed analysis revealed that this inactivation was similar to the O(2)-dependent inactivation of the enzyme in the absence of catecholic substrate, resulting in oxidation of the active site Fe(II) to Fe(III). Interestingly, the catecholic substrate not only increased the reactivity of the enzyme with O(2) to promote ring cleavage but also increased the rate of O(2)-dependent inactivation. Thus, in air-saturated buffer, the apparent rate constant of inactivation of the free enzyme was (0.7 +/- 0.1) x 10(-3) s(-1) versus (3.7 +/- 0.4) x 10(-3) s(-1) for 2,3-dihydroxybiphenyl, the preferred catecholic substrate of the enzyme, and (501 +/- 19) x 10(-3) s(-1) for 3-chlorocatechol, a potent inactivator of 2,3-dihydroxybiphenyl 1,2-dioxygenase (partition coefficient = 8 +/- 2, K(m)(app) = 4.8 +/- 0.7 microm). The 2,3-dihydroxybiphenyl 1,2-dioxygenase-catalyzed cleavage of 3-chlorocatechol yielded predominantly 2-pyrone-6-carboxylic acid and 2-hydroxymuconic acid, consistent with the transient formation of an acyl chloride. However, the enzyme was not covalently modified by this acyl chloride in vitro or in vivo. The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme-catecholic-dioxygen ternary complex and is consistent with the catalytic mechanism.

Burkholderia↗
PubMed2001

Isolation of a benzoate-utilizing Pseudomonas strain from soil and production of catechol from benzoate by transpositional mutants.

Pseudomonas sp. Ba-0511 was isolated from soil by enrichment cultivation on a medium containing 6 mg/ml of sodium benzoate. The bacterium could grow on a medium containing 20 mg/ml of sodium benzoate by a successive enrichment culture. One hundred and twelve transpositional mutants of the bacterium produced catechol from benzoate and accumulated it outside of the cells. Among the mutants, strain BA+63 produced a maximal amount of catechol (2.3 mg/ml) from 6 mg/ml of sodium benzoate after growing for 10.5 h. The conversion rate of benzoate to catechol was 50% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 6 mg/ml of sodium benzoate reached 3.3 mg/ml at the conversion rate of 72% after 5 h of incubation. The resting cells converted m-methylbenzoic acid to 3- and 4-methylcatechol and m-chlorobenzoic acid to 3- and 4-chlorocatechol.

Benzoates↗
PubMed1963

Morphological and pharmacological effects of reserpine, given alone or after iproniazid, on the catechol amines of the adrenal glands of the rat.

Adrenomedullary cells, after fixation with OsO(4), are filled with well formed granules which are considered to represent their catechol amine content. The submicroscopic appearance of these cells was studied in reserpine-treated rats during the late phase of catechol amine depletion and during the period of its restoration. At 3 days after the beginning of reserpine treatment, the granules appeared to be emptied of their content and small vesicles containing scattered, dense deposits of, presumably, catechol amines began to be seen. At 9 days after the beginning of treatment, these deposits had already become granules and the cells had attained a completely normal appearance. The submicroscopic structure of the adrenomedullary cells of rats pretreated with iproniazid (before reserpine), in which a complete inhibition of monoamine oxidase activity had thus been obtained, was similar to that seen in non-treated animals. In numerous cases, however, some characteristic features were noted: the sacs which usually contained a dense granule of catechol amines appeared swollen and many fine granules could be seen around them; the latter were dispersed in a way suggesting that they may represent a partial breakdown of the large granules which, under the inhibitory action of iproniazid, do not release the catechol amines contained within them.

Adrenal Glands↗
PubMed1993

DNA methylation of the pepsinogen 1 gene during rat glandular stomach carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine or catechol.

The methylation patterns of the rat pepsinogen 1 (Pg1) gene in preneoplastic and neoplastic stomach lesions induced by genotoxic N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or the non-genotoxic carcinogen catechol were investigated. Male WKY/Ncrj rats were given MNNG in their drinking water (50 mg/l) for 30 weeks or 0.8% catechol throughout the experiment (60 weeks). MNNG induced Pg1 altered pyloric glands (PAPG), adenomatous hyperplasias and well-differentiated adenocarcinomas. Catechol also induced PAPG and adenomatous hyperplasias although cancers did not develop. Adenomatous hyperplasias and adenocarcinomas all consisted of gastric type cells resembling surface mucous cells or pyloric gland cells with little or no Pg1 expression. In MNNG-induced stomach cancers generally lacking Pg1, altered Pg1 gene methylation was observed with both CCGG and GCGC sites being methylated more than normal pyloric mucosa. MNNG or catechol-induced adenomatous hyperplasias also demonstrated essentially the same methylation changes in the CCGG, but not in the GCGC sites. In the mucosa containing PAPG in groups treated with MNNG or catechol the methylation patterns of the Pg1 gene were quite similar to those of normal pyloric mucosa, although the CCGG sites tended to demonstrate slightly increased methylation. The results suggest that the altered methylation of the Pg1 gene observed in stomach cancers is acquired early in the carcinogenic process and progressive methylation changes occur with tumor development.

Adenocarcinoma↗
PubMed1996

p-Quinone methides are the major decomposition products of catechol estrogen o-quinones.

The mechanism of catechol estrogen-induced carcinogenesis could involve alkylation of critical cellular macromolecules by electrophilic quinoids. The o-quinones formed from peroxidase/P450-catalyzed oxidation of catechol estrogens have previously been implicated as the ultimate carcinogens. In the present study, we have shown that additional reactive intermediates can be produced from isomerization of the catechol estrogen o-quinones to highly electrophilic p-quinone methides (QMs). The o-quinones of the catechol estrogens were incubated at 37 degrees C (pH 7.4) in the absence of GSH. Aliquots were removed at various times and combined with GSH. The GSH adducts were isolated and characterized by 1H-NMR, UV, and electrospray mass spectrometry. The o-quinone of 2-hydroxyestrone isomerized to two QMs; a QM stabilized by one alkyl substituent in the B ring, 2-OHE-QM1 (3-hydroxy-1-(10),3(4),5(6)-oestratrien-2,17-dione) and one having two alkyl substituents on the methylene group in the C ring, 2-OHE-QM2 (2-hydroxy-1(2),4(5),9(10)-oestratrien-3,17-dione). Only one QM was observed from the o-quinone of 4-hydroxyestrone, 4-OHE-QM2 (4-hydroxy-1(2),4(5),9(10)- oestratrien-3,17-dione) which is analogous to the C ring analog (2-OHE-QM2) from the o-quinone of 2-hydroxyestrone. The GSH adduct of 4-OHE-QM2 decomposed at pH 7.4 to give 9(11)-dehydro-4-hydroxyestrone as the major product. Finally, the disappearance of the estrogen o-quinone GSH adducts correlated with the formation of the GSH conjugates of the QMs. These data suggest that in cells with low levels of GSH, the formation of these potent electrophiles represents the major reaction pathway for estrogen o-quinones. The implications of the o-quinone/QM pathway for the in vivo effects of catechol estrogens are not known; however, given the direct link between excessive exposure to endogenous estrogens and the enhanced risk of breast cancer, the potential for formation of additional reactive intermediates needs to be explored.

Estrogens, Catechol↗
PubMed1997

Bioreductive activation of catechol estrogen-ortho-quinones: aromatization of the B ring in 4-hydroxyequilenin markedly alters quinoid formation and reactivity.

There is a clear association between excessive exposure to estrogens and the development of cancer in several tissues including breast and endometrium. The risk factors for women developing these cancers are all associated with longer estrogen exposure, as may be facilitated by early menses, late menopause and long-term estrogen replacement therapy. Equilenin (1,3,5(10),6,8-estrapentaen-3-ol-17-one) or its 17-hydroxylated analogs make up 15% of the most widely prescribed estrogen replacement formulation, Premarin, and yet there is very little information on the human metabolism of these estrogens. In this study, we synthesized the catechol metabolite of equilenin, 4-hydroxyequilenin, and examined how aromatization of the B ring affects the formation and reactivity of the o-quinone (3,5-cyclohexadien-1,2-dione). 4-Hydroxyequilenin-o-quinone is much more redox-active and longer-lived than the endogenous catechol estrone-o-quinones, which suggests that the mechanism(s) of toxicity of the former could be quite different. Interestingly, the rate of reduction of the 4-hydroxyequilenin-o-quinone is increased at least 13-fold in the presence of NAD(P)H:quinone oxidoreductase (DT-diaphorase). Once NADH is consumed however, the catechol auto-oxidized rapidly to the o-quinone. NADH consumption was accompanied by dicumarol-sensitive oxygen uptake both with the purified enzyme and with cytosol from human melanoma cells with high levels of DT-diaphorase activity. P450 reductase and rat liver microsomes also catalyzed NADPH consumption and oxygen uptake. 4-Hydroxyestrone-o-quinone was also rapidly reduced by NAD(P)H; however, this o-quinone does not auto-oxidize and once the o-quinone is reduced the reaction terminates. Including oxidative enzymes in the incubation completes the redox couple and 4-hydroxyestrone-o-quinone behaves like 4-hydroxyequilenin-o-quinone. These data suggest that reduction of estrogen-o-quinones may not result in detoxification. Instead this could represent a cytotoxic mechanism involving consumption of reducing equivalents (NADH/NADPH) as well as formation of superoxide and other reactive oxygen species leading to oxidative stress. Finally, we have compared the cytotoxicity of 4-hydroxyequilenin with that of the estrone catechols in human melanoma cells. 4-Hydroxyequilenin is 5-fold more toxic in these cells compared with 4-hydroxyestrone (ED50 = 7.8 versus 38 microM, respectively) suggesting that formation of the longer-lived redox-active 4-hydroxyequilenin-o-quinone was responsible for the cytotoxic differences. These results substantiate the conclusion that the involvement of quinoids in catechol estrogen toxicity depends on a combination of the rate of formation of the o-quinone, the lifetime of the o-quinone, and the electrophilic/redox reactivity of the quinoids.

Animals↗
PubMed1999

Spinal action of ketorolac, S(+)- and R(-)-ibuprofen on non-noxious activation of the catechol oxidation in the rat locus coeruleus: evidence for a central role of prostaglandins in the strychnine model of allodynia.

BACKGROUND: Blockade of spinal glycine receptors with intrathecal strychnine produces an allodynia-like state in the anesthetized rat. Innocuous hair deflection in the presence of intrathecal strychnine induces a nociceptive-like activation of catechol oxidation in the locus coeruleus and enhances cardiovascular responses. Because prostaglandins play a central role in augmenting pain, this study evaluated the effect of intrathecal nonsteroidal antiinflammatory drugs in strychnine-induced allodynia. METHODS: In urethane-anesthetized rats, changes in catechol oxidation in the locus coeruleus, measured using in vivo voltammetry, and cardiovascular parameters evoked by hair deflection of caudal dermatomes were determined after strychnine (40 microg) or saline were administered intrathecally. Subsequently, the effects of 30 microg ketorolac, 10 microg S(+)-ibuprofen, and 10 microg R(-)-ibuprofen administered intrathecally were evaluated. RESULTS: After strychnine was administered intrathecally, hair deflection evoked an increase in the locus coeruleus catechol oxidation (peak, 149.7+/-7.2% of baseline) and mean arterial blood pressure (peak, 127.5+/-3.8% of baseline). These responses were not observed after saline was administered intrathecally. All hair deflection-evoked, strychnine-dependent peak responses were attenuated significantly with intrathecally administered ketorolac and S(+)-ibuprofen but not with R(-)-ibuprofen. CONCLUSIONS: Locus coeruleus catechol oxidation is a sensitive biochemical index of strychnine-induced allodynia and is correlated temporally with the cardiovascular responses evoked by hair deflection during spinal glycinergic inhibition. The ability of intrathecally administered ketorolac and S(+)-ibuprofen, but not R(-)-ibuprofen, to suppress the locus coeruleus catechol oxidation and cardiovascular peak responses evoked during strychnine-induced allodynia provide evidence that central prostaglandins play an important role in the abnormal sensory processing of strychnine-induced allodynia.

Anesthesia↗