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PubMed1999

Purification of catechol siderophores by boronate affinity chromatography: identification of chrysobactin from Erwinia carotovora subsp. carotovora.

Catechols are co-planar cis-diols known to form stable, isolable complexes with borate under weakly basic conditions. We exploited this chemistry and developed a boronate affinity chromatography for isolating catechol siderophores. The method was applied to the isolation of chrysobactin, enterobactin, and an unknown catechol siderophore produce by Erwinia carotovora subsp. carotovora W3C105. Yields of chrysobactin and enterobactin purified by boronate affinity chromatography were at least two-fold greater than those achieved through alternate methods. The unknown catechol produced by E. carotovora subsp. carotovora W3C105 was isolated by boronate affinity chromatography and shown to be identical to chrysobactin. Boronate affinity chromatography enabled separation of catechol from its rust-colored decomposition products, and simultaneous isolation of catechol and hydroxamate siderophores. Boronate affinity chromatography is a rapid and efficient method for purifying catechol siderophores from bacterial culture supernatants.

Borates↗
PubMed2004

Visible absorption spectra of metal-catecholate and metal-tironate complexes.

Interactions between metals and catechol (1,2-dihydroxybenzene) or other ortho-dihydroxy moieties are being found in an increasing number of biological systems with functions ranging from metal ion internalization to biomaterial synthesis. Although metal-catecholate interactions have been studied in the past, we present the first systematic study of an array of these compounds, all prepared under identical conditions. We report the ultraviolet-visible absorption (UV-vis) spectra for catecholate and tironate complexes of the first row transition elements. Generation and identification of these species were accomplished by preparing aqueous solutions with varied ligand:metal ratios and subsequently titrating with base (NaOH). Controlled ligand deprotonation and metal binding resulted in sequential formation of complexes with one, two, and sometimes three catecholate or tironate ligands bound to a metal ion. We prepared the mono-, bis- and tris-catecholates and -tironates of Fe(3+), V(3+), V(4+)and Mn(3+), the mono- and bis-catecholates and -tironates of Cu(2+), Co(2+), Ni(2+), Zn(2+), Cr(2+) and Mn(2+), and several Ti(4+) and Cr(3+) species. The UV-vis spectra of each complex are described, some of which have not been reported previously. These data can now be applied to characterization of biological metal-catecholate systems.

Catechols↗
PubMed2004

The effect of catechol O-methylation on radical scavenging characteristics of quercetin and luteolin--a mechanistic insight.

The biological effect of flavonoids can be modulated in vivo due to metabolism. The O-methylation of the catechol group in the molecule by catechol O-methyl transferase is one of the important metabolic pathways of flavonoids. In the present study, the consequences of catechol O-methylation for the pH-dependent radical scavenging properties of quercetin and luteolin were characterized both experimentally and theoretically. Comparison of the pKa values to the pH-dependent TEAC profiles reveals that O-methylation not only affects the TEAC as such but also modulates the effect of changing pH on this radical scavenging activity due to an effect on the pKa for deprotonation. The pH-dependent TEAC curves and computer calculated electronic parameters: bond dissociation energy (BDE) and ionisation potential (IP) even indicate that O-methylation of the luteolin catechol group affects the radical scavenging potential only because it shifts the pKa for deprotonation. O-Methylation of the quercetin catechol moiety affects radical scavenging capacity by both an effect on the pKa, and also by an effect on the electron and hydrogen atom donating properties of the neutral (N) and the anionic (A) form of the molecule. Moreover, O-methylation of a catechol OH-group in quercetin and luteolin has a similar effect on their TEAC profiles and on calculated parameters as replacement of the OH-group by a hydrogen atom. Altogether, the results presented provide new mechanistic insight in the effect of catechol O-methylation on the radical scavenging characteristics of quercetin and luteolin.

Antioxidants↗
PubMed2003

Catechol-O-methyltransferase and monoamine oxidase A genotypes and drug response to conventional neuroleptics in schizophrenia.

Biogenic amine synthesis and degradation are involved in the pathogenesis of schizophrenia. Catechol-O-methyltransferase and monoamine oxidase enzymes are important agents in the metabolic inactivation of these neurotransmitters (ie, dopamine, serotonin, and norepinephrine). Functional polymorphism in the catechol-O-methyltransferase and monoamine oxidase A genes causes variation in enzyme activities. We investigated the relationship of catechol-O-methyltransferase Val158Met and monoamine oxidase A promoter repeat polymorphism with response to conventional neuroleptic treatment in schizophrenia.Ninety-four schizophrenic patients formed 2 different study populations. The responders had experienced a fair and steady response to conventional neuroleptics. The nonresponders had failed to achieve an acceptable response to conventional neuroleptics. We also used a control population of 94 age-matched and gender-matched blood donors. Genotyping of the catechol-O-methyltransferase and monoamine oxidase A genes was performed by polymerase chain reaction.Forty-three percent of the nonresponders had a low activity catechol-O-methyltransferase genotype compared with 16% of the responders (P = 0.009). Monoamine oxidase A genotype alone did not differ significantly between the groups. Moreover, the risk of having both low-activity catechol-O-methyltransferase and monoamine oxidase A genotypes was over 6 times more common (odds ratio = 6.16, P = 0.03) in the nonresponders compared with responders. The whole population of patients with schizophrenia did not differ from the controls.The low-activity catechol-O-methyltransferase genotype may be associated with unsatisfactory drug response to conventional neuroleptics or alternatively be involved in a subset of schizophrenics. The role of monoamine oxidase A genotype seems to be additive in this respect.

Adult↗
PubMed2002

Brucella abortus strain 2308 produces brucebactin, a highly efficient catecholic siderophore.

Brucella abortus is known to produce 2,3-dihydroxybenzoate (2,3-DHBA) and to use this catechol as a siderophore to grow under iron-limited conditions. In this study a mutant (BAM41) is described that is deficient in siderophore production by insertion of Tn5 in the virulent B. abortus strain 2308. This mutant was unable to grow on iron-deprived medium and its growth could not be restored by addition of 2,3-DHBA. Production of catecholic compounds by both the Brucella mutant and parental strains under iron-deprivation conditions was assayed by TLC. Two catecholic substances were identified in the supernatant of the parental strain 2308. The faster migrating spot showed the same retention factor (R(f)) as that of purified 2,3-DHBA. The mutant BAM41 overproduced 2,3-DHBA, but failed to form the slower migrating catechol. This defect could only be complemented by the addition of the slow-migrating catechol from strain 2308. The genomic region containing Tn5 in BAM41 was cloned and the position of the transposon was determined by nucleotide sequencing. The sequence revealed that the insertion had occurred at a gene with homology to Escherichia coli entF, a locus involved in the late steps of the biosynthesis of the complex catecholic siderophore enterobactin. Intracellular survival and growth rates of the B. abortus wild-type and entF mutant strains in mouse-derived J774 macrophages were similar, indicating that production of this siderophore was not essential in this model of infection. It is concluded that B. abortus synthesizes a previously unknown and highly efficient catecholic siderophore, different from 2,3-DHBA, for which the name brucebactin is proposed.

Animals↗
PubMed1986

Sites and mechanisms of action of catechol (1,2-dihydroxybenzene) in the rat olfactory cortex slice.

Synaptic transmission in the isolated olfactory cortex slice from the rat was monitored by recording the surface field potentials evoked on lateral olfactory tract (LOT) stimulation. Catechol (approximately 0.05 to 2 mM) caused a concentration-dependent, partially reversible increase in the amplitudes of all field potentials. In a series of conditioning experiments, catechol (1 mM) potentiated postsynaptic inhibition by a mechanism which was at least partially picrotoxin-insensitive. When the relationship between the stimulus input and evoked output was investigated in picrotoxin-treated slices, for a given tract action potential amplitude, catechol (0.25 and 0.5 mM) increased the amplitude of the field potential known as the N-wave; in contrast, for a given N-wave amplitude, the latency of the population spike was increased. Catechol (1 mM) increased the K+-evoked release of endogenous aspartate by a tetrodotoxin-insensitive mechanism whereas the release of glutamate and gamma-aminobutyric acid (GABA) was unaffected. Catechol (1 mM) had no effect on submaximal depolarizations evoked by L-aspartate, L-glutamate or GABA. It is concluded that catechol potentiates excitatory transmission at the LOT-superficial pyramidal cell synapse, possibly by increasing evoked transmitter release. Other synaptic actions of catechol may be consequent upon this increased excitatory input but the results do not exclude the possibility of separate and distinct actions on polysynaptic transmission.

Action Potentials↗
PubMed1986

Catechol: a potent and specific inhibitor of the fast potassium channel in frog primary afferent neurones.

The effects of catechol on various ionic channels of isolated primary afferent neurones of the bull-frog were examined by a single-suction-electrode clamp system, which combined internal perfusion and current or voltage clamp using an electronic switching circuit. Catechol was found to inhibit rather specifically the fast K+ current as does 4-aminopyridine (4-AP). Ca2+, Na+ and slow K+ currents were not affected. Although both 4-AP and catechol were inhibitors of the fast K+ channels, their sites of action were quite different. Catechol was effective when applied on the external surface of the cell membrane whereas 4-AP acted preferably internally. We assumed that a single fast K+ channel has two distinct sites for blockers: the catechol site is exposed to the external medium or situated at the outer orifice of the pore, and the 4-AP site is located within the same channel but is more easily accessible from inside the nerve cell than outside. The 4-AP and catechol sites were not, however, completely separate and independent of each other since a synergistic interaction was observed between catechol and 4-AP.

4-Aminopyridine↗
PubMed2004

Oxidation of benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1.

Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 microM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 +/- 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 +/- 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 +/- 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 +/- 1, 3.1 +/- 0.3, and 0.26 +/- 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 +/- 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 +/- 0.07 and 1.5 +/- 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 +/- 0.8, 4.0 +/- 0.6, and 2.4 +/- 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 microM).

Base Sequence↗
PubMedDate not supplied

Sequential morphological and biological changes in the glandular stomach induced by oral administration of catechol to male F344 rats.

Histogenesis and mechanisms of catechol-induced rat glandular stomach carcinogenesis were investigated in male F344 rats. Groups of 5 or 6 rats were treated with dietary catechol at doses of 1, 0.5, 0.1, and 0.01% for 12 hr or for 1, 2, 3, or 7 days or at a dose of 0.8% for 1, 2, 4, 12, and 24 wk; rats were then euthanatized. The initial morphological changes were edema of the gastric wall, inflammatory-cell infiltration, erosion in the pyloric region close to the duodenum, and considerable increase in apoptosis at 12 hr; later, changes included augmented DNA synthesis and cell proliferation, as evaluated by bromodeoxyuridine labeling index and thickness of mucosa, respectively, on day 1. Downward hyperplasia due to excess regeneration appeared at edges of ulceration at week 2. This lesion disappeared, and then submucosal hyperplasia appeared in the course of adenoma development. Only slight expression of c-myc or c-fos was apparent after 30-min oral administration or 1-, 3-, and 6-hr oral administration of catechol. No increase in lipid peroxide levels was evident in gastric epithelium fed catechol for 1 wk. The amount of catechol distributed in the glandular stomach and forestomach epithelium, which is not a target for carcinogenesis, did not differ 1, 3, 6, and 24 hr after a single intragastric dose of 75 mg/kg body weight. Amounts of catechol bound to tissue protein were also not specifically high in the glandular stomach. These results indicate that regenerative cell proliferation due to toxicity plays an important role in catechol-induced glandular stomach carcinogenesis. Protein binding and free radicals may not be largely responsible for the toxicity.

Administration, Oral↗
PubMed1986

Inhibition of the preovulatory prolactin surge in the rat by catechol estrogens: functional and temporal specificity.

Catechol estrogens, administered iv to cycling rats on the morning of proestrus, were able to block the preovulatory PRL surge on the afternoon of the same day. Only catechol estrogens with a low affinity for the estrogen receptor, such as 2-hydroxyestrone and 2-hydroxyestradiol-17 alpha were effective in this respect, while the estrogenic catechol estrogen 2-hydroxyestradiol was unable to influence the PRL surge. The effectiveness of the PRL surge abolition was highly dependent on the state of the endogenous estradiol levels at the time of administration. Only doses given just before the peak secretion of estradiol were effective in blocking the PRL surge. Despite similarities in the inhibition of the preovulatory LH and PRL surges by catechol estrogens, these are considered to occur by different mechanisms because the LH secretion is blocked by all catechol estrogens, while the PRL surge is affected only by catechol estrogens with no estrogen agonist properties. The catechol estrogen blockade of the PRL surge may have physiological parallels and provides a useful probe of the mechanisms of the estrogen-PRL axis.

Animals↗
PubMed1995

On the inhibitory effect of C17-sulfoconjugated catechol estrogens upon lipid peroxidation of rat liver microsomes.

The antioxidant effect of C17-sulfoconjugated catechol estrogens was examined under ascorbic acid- or NADPH-dependent lipid peroxidation in rat liver microsomes and compared with that of various estrogens and alpha-tocopherol. Among the estrogens tested, a free catechol estrogen such as 4-hydroxyestradiol showed the strongest effect, followed by 2-hydroxyestradiol, 2-methoxyestradiol and estradiol. Next to these steroids, 2-hydroxyestradiol 17-sulfate, followed by 4-methoxyestradiol, 4-hydroxyestradiol 17-sulfate and estrone also showed a strong inhibitory effect, which was greater than that of alpha-tocopherol. Among the C17-sulfates, the guaiacols (2- and 4-methoxyestradiol 17-sulfate) showed a slightly lower effect than alpha-tocopherol, but estradiol 17-sulfate had almost no effect. The antioxidant activity observed in phenolic or guaiacol steroids was considered to be attributed to the catechols produced by their 2- (or 4-)hydroxylation or their O-demethylation, respectively, during the incubation. This was confirmed by identification of the catechols produced from phenolic or guaiacol estrogens and even from the estrogen C3-sulfates. The mechanism of the inhibition by catechols on lipid peroxidation was speculated to involve their activity as radical scavengers, because of their strong reducing activity for 1,1-diphenyl-2-picrylhydrazyl. The above results suggest that C17-sulfoconjugated catechol estrogens (2- and 4-hydroxyestradiol 17-sulfate), although with slightly lower activity than their free catechols, are promising endogenous antioxidants. The physiological role of these estrogen conjugates during pregnancy is discussed.

Animals↗
PubMed1986

Regioselectivity of catechol O-methyltransferase. The effect of pH on the site of O-methylation of fluorinated norepinephrines.

Selectivity of catechol O-methyltransferase has been examined for the three ring-fluorinated norepinephrines to elucidate the role of acidity of the phenolic groups in their methylation. Substitution of fluorine at the 5-position of norepinephrine reverses the selectivity of catechol O-methyltransferase so that p-O-methylation predominates. The 5-fluoro substituent also causes the pKa of the p-hydroxyl group to decrease substantially. In contrast, 2- and 6-fluoronorepinephrines are methylated predominantly at the m-hydroxyl group. These results suggest that acidity of a phenolic group can play an important role in its ability to be methylated by catechol O-methyltransferase. Percentages of p-O-methylation of norepinephrine and its fluorinated derivatives increase with pH. This relative increase in p-O-methylation appears to accompany ionization of a group with pKa of 8.6, 7.7, 7.9, and 8.4 for norepinephrine and its 2-, 5-, and 6-fluoro derivative, respectively. These pKa values are the same as or similar to the pKa values of a phenolic hydroxyl group of these substrates. 3,4-Dihydroxybenzyl alcohol and its 5-fluoro derivative are O-methylated by catechol O-methyltransferase to form p- and m-O-methyl products in approximately 1:1 and 4:1 ratios, respectively, at all pH values. Based on the above results, a catechol-binding site model for catechol O-methyltransferase is proposed in which the two phenolic hydroxyl groups of catechol substrates are postulated to be approximately equally spaced from the methyl group of the cosubstrate S-adenosylmethionine.

Binding Sites↗
PubMed1982

Enzymatic O-methylation of catechols and catecholamines.

The reactivity of a number of catechols and catecholamines with regard to the enzymatic O-methylation by catechol-O-methyltransferase (COMT) was studied. The reaction was carried out in a vial at a temperature of 37 degrees C, the vial contained a certain concentration of one catechol(amine), catechol-O-methyltransferase (the enzyme), S-adenosylmethionine (the methyldonor), MgCl2 (a cofactor) and buffer pH = 7.85. After certain time intervals samples were taken, the reaction was stopped in acid. The catechol(amine) concentration decrease and the product concentration increase were determined by injecting the samples directly into an HPLC connected with a fluorimeter, giving the opportunity of estimating the mass balance. Vmax, Km, C3/C4 ratio (= ratio of 3-O- and 4-O-methylated product formed) and the reaction rate at low substrate (catecholamine) concentration (= Vmax. [S]/Km)-which appears to be related to log P - are given. It is conjectured that V at low substrate concentration is especially determined by the polarity of the catechol(amine) while Vmax is primarily determined by other physico-chemical properties like steric conformation (L-dopa vs DL-dopa; L-adrenaline vs DL-adrenaline).

Catechol O-Methyltransferase↗
PubMed2004

Mechanistic insight into the catechol oxidase activity by a biomimetic dinuclear copper complex.

The biomimetic catalytic oxidation of 3,5-di- tert-butylcatechol by the dicopper(II) complex of the ligand alpha,alpha'-bis(bis[1-(1'-methyl-2'-benzimidazolyl)methyl]amino)- m-xylene in the presence of dioxygen has been investigated as a function of temperature and pH in a mixed aqueous/organic solvent. The catalytic cycle occurs in two steps, the first step being faster than the second step. In the first step, one molecule of catechol is oxidized by the dicopper(II) complex, and the copper(II) centers are reduced. From the pH dependence, it is deduced that the active species of the process is the monohydroxo form of the dinuclear complex. In the second step, the second molecule of catechol is oxidized by the dicopper(I)-dioxygen complex formed upon oxygenation of the reduced complex. In both cases, catechol oxidation is an inner-sphere electron transfer process involving binding of the catechol to the active species. The binary catechol-dicopper(II) complex formed in the first step could be characterized at very low temperature (-90 degrees C), where substrate oxidation is blocked. On the contrary, the ternary complex of dicopper(I)-O(2)-catechol relevant to the second step does not accumulate in solution and could not be characterized, even at low temperature. The investigation of the biphasic kinetics of the catalytic reaction over a range of temperatures allowed the thermodynamic (Delta H degrees and Delta S degrees ) and activation parameters (Delta H( not equal) and Delta S( not equal)) connected with the key steps of the catecholase process to be obtained.

Biomimetic Materials↗
PubMed2005

Spectroscopic studies of the anaerobic enzyme-substrate complex of catechol 1,2-dioxygenase.

The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme/substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UV-vis) difference spectroscopic studies demonstrated that, in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that, in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm(-1) in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm(-1) in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution.

Anaerobiosis↗
PubMed1994

Substrate specificity of catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida and its relationship to cell growth.

Catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida consists of four identical subunits, each containing one ferrous ion. The enzyme catalyzes ring cleavage of catechol, 3-methylcatechol, and 4-methylcatechol but shows only weak activity toward 4-ethylcatechol. Two mutants of catechol 2,3-dioxygenases (4ECR1 and 4ECR6) able to oxidize 4-ethylcatechol, one mutant (3MCS) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave catechol and 4-methylcatechol, and one phenotypic revertant of 3MCS (3MCR) which had regained the ability to oxidize 3-methylcatechol were characterized by determining their Km and partition ratio (the ratio of productive catalysis to suicide catalysis). The amino acid substitutions in the four mutant enzymes were also identified by sequencing their structural genes. Wild-type catechol 2,3-dioxygenase was inactivated during the catalysis of 4-ethylcatechol and thus had a low partition ratio for this substrate, whereas the two mutant enzymes, 4ECR1 and 4ECR6, had higher partition ratios for it. Similarly, mutant enzyme 3MCS had a lower partition ratio for 3-methylcatechol than that of 3MCR. Molecular oxygen was required for the inactivation of the wild-type enzyme by 4-ethylcatechol and of 3MCS by 3-methylcatechol, and the inactivated enzymes could be reactivated by incubation with FeSO4 plus ascorbic acid. The enzyme inactivation is thus most likely mechanism based and occurred principally by oxidation and/or removal of the ferrous ion in the catalytic center. In general, partition ratios for catechols lower than 18,000 did not support bacterial growth. A possible meaning of the critical value of the partition ratio is discussed.

Ascorbic Acid↗
PubMed1996

Evidence that the catechol 3,4-Dihydroxytamoxifen is a proximate intermediate to the reactive species binding covalently to proteins.

Metabolism of tamoxifen by rat and human hepatic microsomal cytochrome P450s (CYPs) forms a reactive intermediate that irreversibly binds to microsomal proteins (C. Mani and D. Kupfer, Cancer Res., 51: 6052-6058, 1991.). The current study examines the nature of the tamoxifen metabolite that is proximate to the reactive intermediate(s). The rate of covalent binding of tamoxifen metabolites, tamoxifen N-oxide, N-desmethyltamoxifen, and tamoxifen N-oxide-epoxide was approximately equal to or less than that of tamoxifen. By contrast, covalent binding of 4-hydroxytamoxifen (4-OH-tam) was 3-5-fold higher than that of tamoxifen, indicating that among the metabolites examined, 4-OH-tam or its metabolite(s) is most proximate to the reactive intermediate(s). Incubation of 4-OH-tam with liver microsomes from PCN-treated rat yielded three detectable metabolites. One was identified as 4-OH-tam N-oxide via its facile reduction back to 4-OH-tam by titanium(III) chloride. Another metabolite of 4-OH-tam, assumed to be 3,4-dihydroxytamoxifen (3,4-di-OH-tam) catechol, was demonstrated by its monomethylation with [3H]S-adenosyl-L-methionine ([3H]SAM) in presence of endogenous catechol-O-methyltransferase. Monomethylated catechol from 4-OH-tam was formed at a 3-4-fold higher rate than from tamoxifen. It was reasoned that if the catechol is most proximate metabolite to the reactive intermediate, then its methylation would reduce the formation of the reactive intermediate and result in lower rate of covalent binding. In fact, addition of radioinert SAM to incubations of tamoxifen inhibited covalent binding by 17-23%. By contrast, inclusion of 1.0 mM S-adenosyl-L-homocysteine, a potent inhibitor of catechol-O-methyltransferase-mediated methylation of 3,4-di-OH-tam, essentially overcame the inhibition of the covalent binding by SAM. Additionally, ascorbic acid and glutathione, inhibitors of covalent binding of tamoxifen, produced an elevation of methylated catechol. These findings collectively indicate that 3,4-di-OH-tam is proximate to the ultimate reactive intermediate that results in covalent binding to microsomal proteins.

Animals↗
PubMed1991

A method for estimating catechol estrogen metabolism from excretion of noncatechol estrogens.

The relationship of catechol estrogen metabolism to disease has seldom been investigated because of analytic difficulties. Estradiol (E2) and estrone (E1) are oxidized simultaneously at either ring A or ring D, and the rate of catechol estrogen formation (r2) is reciprocally related to the rate of 16 alpha-hydroxylation (r3). The rate of ovarian estrogen production (X10) can be summarized as to metabolic outcome: X10 = r10 + r2 + r3 + r(u), where r10 is the loss of E1 and E2 in urine, and ru is the fecal and urinary loss of unknown oxidative products. Assuming a constant r(u) between subjects: constancy of the X10 concentration between subjects during similar menstrual cycle phases. In the absence of xenobiotics, r2 x r3 are reciprocally interrelated: r2 x r3 = K (an oxidation constant whose limiting factor is the biologically available estrogen at the cell surface). To the extent that r10 approximates estrogens available for cellular metabolism, the rate of catechol estrogen metabolism may be determined from (Formula; see text) From published data K = 12.4 +/- 0.8 of the standard error of the mean. Pearson correlation coefficients between actual and estimated catechol estrogen excretion in groups of subjects ranged from 0.61 to 0.97 (median, 0.88). This method has been useful for clinical investigation of the relationship of catechol estrogen metabolism to disease until better methods to measure catechol estrogen directly are available.

Body Weight↗