Biomedical subjects

D R Doerge

Publications and source records attributed to D R Doerge.

At least 55 records · Page 3Linked to original sources

PubMed1997

Quantification of heterocyclic amine carcinogens in cooked meats using isotope dilution liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry.

Heterocyclic aromatic amines (HAAs) are formed in cooked meats through pyrolysis reactions of different amino acids in the presence or absence of creatine/creatinine and sugars. HAAs are mutagens, colon/mammary gland carcinogens in rodents, and are suspected in the etiology of human cancers. In this study, cooked meats containing incurred HAAs as well as control (microwave) meat, were spiked with four labeled HAA internal standards (MeIQx, IQ, AAC and PhIP) and extracted using a liquid/liquid cleanup procedure. Isotope dilution measurements were made using on-line liquid chromatography atmosphere pressure chemical ionization tandem mass spectrometry with multiple reaction monitoring to provide the sensitivity and specificity needed for trace analysis in these complex matrices. The procedure was validated using control meat spiked with the four native HAAs at 0-50 ppb. The levels of HAAs found in cooked meats ranged from non-detectable (limit of detection 0.1-1.0 ppb) in microwave-cooked hamburger to 226 ppb PhIP and 104 ppb AAC in well-done grilled chicken. This methodology has the potential to provide accurate data on the consumption of HAAs in the diet for use in human cancer risk assessment.

Amines↗

PubMed1997

Metabolism of metolachlor by the fungus Cunninghamella elegans.

The metabolism of metolachlor[2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-met hyl ethyl)acetamide]by the fungus Cunninghamella elegans ATCC 36112 was determined. Thesix metabolites identified comprised 81% of the total[14C]-metolachlor metabolized by C. elegans. Thesemetabolites were separated by reversed-phase high-performance liquidchromatography and identified by 1H nuclear magnetic resonance, UV, and atmospheric pressure chemical ionization (APCI) mass spectraltechniques. Metabolites I and II were identified as stereoismers of2-chloro-N-[2-ethyl-6-hydroxymethylphenyl)]-N-(2-hydroxy-1-me thylet hyl)acetamide. Metabolites III and IV have been tentatively identified as stereoismers of2-chloro-N-[2-(1-hydroxyethyl)-6-methylphenyl]-N-(2-methoxy-1-++ +methy lethyl)acetamide. Metabolites V and VI were identified as stereoismers of2-chloro-N-(2-ethyl-6-hydroxy-methylphenyl)-N-(2-methoxy-1-me thylet hyl)acetamideand 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-hydroxy-1-methylethyl)acetam ide, respectively. The fungus Cunninghamellaelegans was able to biotransform metolachlor. Multiple site oxidation ofmetolachlor by C. elegans occurred predominantly byO-demethylation of the N-alkyl side chain and benzylichydroxylation of the arylalkyl side chain.

Acetamides↗

PubMed1997

Mechanism for the anti-thyroid action of minocycline.

Administration of minocycline (MN), a tetracycline antibiotic, produces a black pigment in the thyroids of humans and several species of experimental animals and antithyroid effects in rodents. We have previously shown that these effects appear to be related to interactions of MN with thyroid peroxidase (TPO), the key enzyme in thyroid hormone synthesis. In the present study, the mechanisms for inhibition of TPO-catalyzed iodination and coupling reactions by MN were investigated. MN was stable in the presence of TPO and H2O2, but adding iodide or a phenolic cosubstrate caused rapid conversion to several products. TPO-dependent product formation, characterized by on-line LC-APCI/MS and 1H-NMR, involved oxidative elimination to form the corresponding benzoquinone with subsequent dehydrogenation at the aliphatic 4-(dimethylamino) group. Addition of thiol-containing polymers (bovine serum albumin or thiol-agarose chromatographic beads) had a minimal effect on MN oxidation by TPO, but substantially reduced product formation and produced concomitant losses in free thiols. Covalent bonding through a thioether linkage of a reactive intermediate, the benzoquinone iminium ion, was inferred from these findings. Iodide- and phenolic cosubstrate-dependent oxidation of tetracycline to demethylated and dehydrogenated products was also observed, although at a slower rate than MN. The products and kinetics observed with MN were consistent with oxidation of MN by either the enzymatic iodinating species formed by reaction of TPO compound I with iodide or phenoxyl radicals/cations generated by TPO-mediated oxidation of a phenolic cosubstrate. The proposed reaction mechanism is consistent with alternate substrate inhibition of TPO-catalyzed iodination of tyrosyl residues in thyroglobulin (Tg) by MN, as previously reported. Furthermore, the observed phenoxyl radical-mediated oxidation of MN is consistent with its previously reported potent inhibition of the coupling of hormonogenic iodotyrosine residues in Tg in the reaction that forms thyroid hormones. The proposed reaction mechanism also implicates a reactive benzoquinone iminium ion intermediate that could be important in toxicity of MN.

Anti-Bacterial Agents↗

PubMed1997

Synthesis and 32P-postlabeling/high-performance liquid chromatography separation of diastereomeric 1,N2-(1,3-propano)-2'-deoxyguanosine 3'-phosphate adducts formed from 4-hydroxy-2-nonenal.

4-Hydroxy-2-nonenal (HNE), a major electrophilic byproduct of lipid peroxidation, is mutagenic and cytotoxic. The two pairs of HNE-derived diastereomeric 1,N2-propanodeoxyguanosine 3'-monophosphate adducts were synthesized from reaction of HNE with 2'-deoxyguanosine 3'-monophosphate. After HPLC separation, these adducts were characterized by UV-visible absorption and negative ion electrospray ionization MS/MS analysis. To further characterize the structures, these adducts were dephosphorylated to the corresponding HNE-modified deoxyguanosine adducts and their HPLC retention times and UV spectra were compared with those of the synthetic standards prepared from reaction of HNE with 2'-deoxyguanosine. Separation of these adducts by 32P-postlabeling/HPLC was developed. Reaction of HNE with calf thymus DNA resulted in only one pair of diastereomeric adducts, with one adduct predominantly formed with a modification level of 1.2 +/- 0.5 adducts/10(7) nucleotides.

Aldehydes↗

PubMed1996

Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase.

Thyroid peroxidase (TPO) simultaneously catalyzes two very different types of reaction in the thyroid gland- iodination and coupling. The present study addresses the mechanism of this simultaneous dual activity. Compound I, the two-electron oxidation product of TPO, exists in two different forms--an oxoferryl porphyrin pi-cation radical and an oxoferryl protein radical. It has been proposed that iodination is mediated by the porphyrin pi-cation radical form of TPO compound I, while coupling is mediated by the protein radical form. However, results obtained in the present study favor the view that both iodination and coupling are mediated by the porphyrin pi-cation radical form of compound I. In the first part of the study, we compared coupling and iodination activities of two peroxidases with very similar crystal structures--cytochrome c peroxidase (CcP) and lignin peroxidase (LiP). Although these two peroxidases have very similar three-dimensional structures, CcP forms a compound I only of the protein radical type, whereas compound I of LiP exists only as a porphyrin pi-cation radical. Comparison of the catalytic activities of the two enzymes showed that diiodotyrosine (DIT)-stimulated coupling activity of LiP was significantly greater than that of CcP. Moreover, lignin peroxidase displayed very significant iodinating activity at acid pHs, whereas iodination with CcP was negligible at all pHs tested. Our findings with these two structurally similar peroxidases suggested that TPO-catalyzed iodination and coupling could both be mediated by the porphyrin pi-cation radical form of compound I. More direct evidence in support of this view was obtained in the second part of this study, employing TPO and lactoperoxidase (LPO) model systems in which iodination and coupling occurred simultaneously. Heme spectral analysis was used to correlate formation of the protein radical form of compound I with the kinetics of the iodination and coupling reactions. Formation of the compound I protein radical was not observed until the iodination and coupling reactions had almost been completed. In separate experiments it was shown that the spontaneous conversion of the porphyrin pi-cation radical form of TPO or LPO compound I to the protein radical form was markedly inhibited by a low concentration of iodide, especially in the presence of an iodide acceptor. These studies provide compelling evidence that both iodination and coupling are mediated by the porphyrin pi-cation radical form of compound I. This was further substantiated by the finding that coupling was inhibited in the presence of excess iodide, an observation readily explained by competition between iodide and DIT residues in thyroglobulin for oxidation by the porphyrin pi-cation radical.

Animals↗

PubMed1996

Detection and confirmation of beta-agonists in bovine retina using LC/APCI-MS.

Beta-adrenergic receptor agonists are growth-promoting drugs with the potential for illegal use in livestock, and human toxicity has resulted from consumption of contaminated meat. On-line liquid chromatography with atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) was used for sensitive detection of several beta-agonists in retina, a tissue reported to concentrate and retain such residues for extended periods. Multiresidue extraction, separation, detection, and confirmation procedures were developed for retinal tissue and applied to eyes from cattle treated with clenbuterol (69-201 ppb) and to control eyes spiked with salbutamol (100 ppb) and terbutaline (25-100 ppb). Rapid switching of the potential difference between sampling cone and skimmer in the transport region of the API source was used to optimize acquisition of the protonated molecules and characteristic fragment ions obtained by collision-induced dissociation reactions. The respective selected ions were simultaneously acquired using a single quadrupole mass spectrometer. The accurate and precise agreement observed for diagnostic ion intensity ratios between beta-agonists in retinal samples and authentic standards suggests that LC/APCI-MS can be used for confirmation of analyte structure at trace levels and does not require the use of a triple-stage quadrupole mass analyzer.

Adrenergic beta-Agonists↗

PubMed1996

Confirmation of gentian violet and its metabolite leucogentian violet in catfish muscle using liquid chromatography combined with atmospheric pressure ionization mass spectrometry.

Gentian violet (GV) is a triphenylmethane dye antiseptic with potential for illegal use in livestock production, especially aquaculture where the related malachite green has been widely used. This potential misuse has regulatory importance because of the observed rodent carcinogenicity of GV. This report describes the use of online LC-APCI/MS for confirmation of incurred GV residues, and those of its principal metabolite, LGV, in catfish muscle following treatment of live catfish with GV under putative use conditions. LC with APCI/MS detection provided sensitive analysis of GV and LGV with estimated detection limits of < 1 pg observed for both compounds. Fragmentation of GV and LGV via in-source CID was effected by varying the sampling cone-skimmer voltage. Ion intensity data were collected using a rapid cone voltage switching procedure that permits selected ion acquisition under optimal conditions for the parent molecule and several selected fragment ions. For GV, four ions including the ionized molecule were used and for LGV, six ions including the protonated molecule were used. The levels of GV and LGV in muscle from fish dosed with 10 micrograms/l in aquarium water were determined by LC/VIS to be 0.5 and 44 ppb, respectively. Analysis of these samples yielded ion intensity ratios that agreed precisely between injections (< 5%) and accurately with those generated by a comparable amount of authentic GV and LGV (< 10% deviation). These results show the utility of on-line LC-APCI/MS to do both sensitive confirmatory analyses of incurred drug residues for use in monitoring the food supply.

Animals↗

PubMed1996

Minocycline and the thyroid: antithyroid effects of the drug, and the role of thyroid peroxidase in minocycline-induced black pigmentation of the gland.

Minocycline (MN), a member of the tetracycline family of antibiotics, is known to induce a black discoloration of the thyroid in several species, including humans. Antithyroid effects of MN have also been reported. The aim of the present study was two-fold: (1) to determine whether thyroid peroxidase (TPO) is involved in the MN-induced black thyroid, and (2) to obtain information on the effect of MN on TPO-catalyzed iodination and coupling in model systems containing highly purified TPO. Treatment of MN with TPO in the presence of the H2O2 generating system, glucose-glucose oxidase, resulted in the formation of a black product (or products). In phosphate buffer, pH 7.0, the color intensity reached its peak in about 90 min. Control samples without TPO showed little or no color change during this interval. Formation of the black product(s) did not require the presence of iodide. Other members of the tetracycline family were not oxidized to dark products by the TPO system. These results provide definitive evidence that TPO is involved in the MN-induced black thyroid. MN is an inhibitor of TPO-catalyzed iodination in model systems, with a potency comparable to that of MMI and PTU. At low drug concentrations (approximately 25 microM), MN appeared to act as a competitive inhibitor, as previously shown for lower concentrations of MMI and PTU. However, when the drug concentration was increased, MN and the thioureylene drugs inhibited iodination by different mechanisms. With PTU and MMI, iodination was irreversibly inhibited through inactivation of TPO. However, inhibition of iodination by MN (100 microM) was not associated with inactivation of TPO and was at least partially reversible. The most potent inhibitory effect of MN was on TPO-catalyzed coupling. This was demonstrated both in a coupling test system, designed to measure coupling in the absence of iodination, and in an iodination system, in which iodination and coupling occurred simultaneously. In both systems, MN was several times more potent than PTU and MMI, or other tetracycline drugs. Based on the potent antithyroid effects of MN observed in our in vitro studies, it seems advisable to monitor thyroid function in patients receiving long-term MN therapy.

Animals↗

PubMed1995

Multiresidue determination of quinolone antibiotics using liquid chromatography coupled to atmospheric-pressure chemical ionization mass spectrometry and tandem mass spectrometry.

Liquid chromatography coupled to atmospheric-pressure chemical ionization mass spectrometry and tandem mass spectrometry (MS/MS) methods for the determination and quantification of four quinolone antibiotics were adapted from a published procedure for liquid-liquid extraction from catfish muscle and high-performance liquid chromatographic analysis. In-source collision-induced dissociation was used to optimize fragmentation to produce mass spectra consisting of the protonated molecule and two characteristic fragment ions of nearly equal intensity. Selected ion monitoring of three ions per quinolone yielded sensitive detection in catfish muscle extracts (estimated instrumental detection limits 0.8-1.7 ppb). The intensity ratios were used to confirm the presence of three of the quinolones based on the accurate agreement (< or equal to 10% deviation) between ratios derived from fortified catfish extracts and those from standard quinolones. MS/MS was used to increase the specificity and sensitivity of analysis. Scans using constant neural loss (CNL) of 18 mass units gave a sensitive response for the quinolones suggesting that CNL scans may be applicable to multiresidue screening for unknown quinolones. MS/MS with multiple reaction monitoring of the [MH - 18]+ transition for each quinolone yielded the highest sensitivity analysis in catfish extracts (estimated instrumental detection limits 0.08-0.16 ppb).

4-Quinolones↗

PubMed1995

Porphyrin pi-cation and protein radicals in peroxidase catalysis and inhibition by anti-thyroid chemicals.

1. Thyroid peroxidase (TPO) catalyses the iodination and phenolic coupling reactions in the biosynthesis of thyroid hormones. 2. The two-electron oxidation of TPO by H2O2 produces an oxoferryl porphyrin pi-cation radical compound I that isomerizes spontaneously to a form of compound I that contains an oxoferryl haem and the second oxidizing equivalent as an amino acid radical. 3. The pi-cation radical compound I is the catalytic species that effects iodide ion oxidation and the protein radical compound I is most likely the catalytic species that catalyses coupling. 4. Methimazole, a therapeutic, anti-hyperthyroid drug, is a suicide substrate for TPO and effects irreversible inactivation by TPO-mediated S-oxygenation to a reactive sulphenic acid that binds covalently to the prosthetic haem. 5. Sulphamethazine and other arylamines containing electron-withdrawing substituents inhibit TPO compound I-mediated reactions by reversible, mixed-type inhibition. 6. Ethylenethiourea, a fungicide metabolite, blocks TPO-mediated iodination by reacting with the catalytic iodinating species as an alternate substrate. 7. Resorcinol and related dietary flavonoids are suicide substrates for TPO and act by covalent binding to amino acid residues, presumably those radical sites present in the compound I isomer. 8. Nitrosobenzene, a known radical-trapping agent, blocks TPO-mediated coupling but not iodination or phenolic oxidations presumably by interception of the 3,5-diiodotyrosyl radical species generated during the coupling reaction.

Animals↗

PubMed1994

Evidence for a radical mechanism in peroxidase-catalyzed coupling. I. Steady-state experiments with various peroxidases.

Biosynthesis of thyroxine in the thyroid gland involves a reaction between two diiodotyrosyl residues within the same molecule of thyroglobulin, a large, thyroid-specific glycoprotein. This reaction, generally referred to as the coupling reaction, is catalyzed in the thyroid by the heme-containing glycoprotein enzyme, thyroid peroxidase, also a thyroid-specific protein. The coupling reaction is, however, not specific for thyroid peroxidase; it is also efficiently catalyzed by other heme-containing peroxidases. Peroxidase-catalyzed coupling may also occur between a monoiodotyrosyl and a diiodotyrosyl residue in thyroglobulin to form the more potent thyroid hormone, 3',3,5-triiodothyronine. Under most conditions, thyroxine formation in the thyroid is greatly favored over that of 3',3,5-triiodothyronine. Two mechanisms have been proposed for the coupling reaction, a radical mechanism and an ionic mechanism. In this, and in the following paper, we present evidence favoring a radical mechanism. This view is bsed primarily on the observation that peroxidase-catalyzed coupling is markedly stimulated by substoichiometric concentrations of free diiodotyrosine (DIT). Evidence obtained in this and in the following paper leads us to conclude that the stimulatory effect of DIT on coupling involves peroxidase-catalyzed oxidation of the added DIT to a radical form. We propose that this stimulation involves a radical chain propagation mechanism. This implies that peroxidase-catalyzed coupling in the absence of DIT must also be a radical-mediated reaction.

Animals↗

PubMed1994

Evidence for a radical mechanism in peroxidase-catalyzed coupling. II. Single turnover experiments with horseradish peroxidase.

Single turnover experiments were performed with horseradish peroxidase (HRP) to study the mechanism of peroxidase-catalyzed coupling and its stimulation by low concentrations of free diiodotyrosine (DIT). HRP was used because, unlike thyroid peroxidase (TPO) and lactoperoxidase (LPO), the spectral properties of compounds I and II are readily distinguishable. This made it possible to correlate the kinetics and stoichiometry of T4 + T3 formation with spectral data. Incubation of 2 microM preformed HRP-I with 2 microM [125I]Tg (thyroglobulin of low hormone content, high iodotyrosine content) in the presence of 1 microM free DIT yielded about 0.8 residue T4 and 0.2 residue T3 per molecule of Tg. This represents the theoretical maximum for iodothyronine formation, indicating remarkably efficient use of the oxidizing equivalents in HRP-I for coupling. The time course for formation of T4 + T3 was biphasic. During a rapid initial phase (about 1 min), HRP-I was completely converted to HRP-II, coincident with the formation of about 0.65 residues of T4 + T3. During the second slower phase, lasting 10-15 min, HRP-II was completely reduced to the native enzyme, with formation of the remaining T4 + T3. In the absence of DIT, the coupling yield was reduced to 0.5-0.6 residue T4 + T3 per molecule Tg, and the reaction, although considerably slower, was still biphasic. The rapid phase again corresponded to the conversion of HRP-I to HRP-II, and the slower phase to the conversion of HRP-II to native enzyme. To gain insight into the mechanism of the stimulatory effect of free DIT on coupling, we studied the reaction of DIT with HRP-I and HRP-II. Free DIT reacted with both HRP-I and HRP-II in one-electron transfer reactions, and the time course for these reductions resembled those observed with DIT + Tg. These observations suggest that in DIT-stimulated coupling, free DIT radicals act as a shuttle for transferring oxidizing equivalents from the peroxidase intermediates to the DIT residues in Tg. The remarkable efficiency of the HRP-I-mediated coupling reaction implies that (i) only hormonogenic residues in Tg are oxidized and (ii) oxidation of two hormonogenic residues occurs within the same molecule of Tg. A scheme which attempts to explain both kinetic and stoichiometric features of the coupling reaction observed in this study is proposed. This scheme is based on a radical mechanism, consistent with the conclusions reached in the companion paper.

Anaerobiosis↗

PubMed1994

Mechanism-based inactivation of lactoperoxidase and thyroid peroxidase by resorcinol derivatives.

Humans are exposed to resorcinol derivatives in the environment through ground water, foods, food additives, drugs, and hair dyes. Epidemiological studies have linked human exposure to phenolic compounds with the thyroid disorder, goiter. The results presented here demonstrate the suicide (mechanism-based) inactivation of thyroid peroxidase (TPO) and the closely related lactoperoxidase (LPO) by resorcinol derivatives. The evidence for this mechanism includes irreversible, hydrogen peroxide-dependent loss of enzymatic activity by kinetics consistent with a suicide mechanism, concomitant with changes in the visible spectrum of the prosthetic heme group and covalent binding of resorcinol (ca. 10 mol/mol of lactoperoxidase inactivated). The inactivation was specific for thyroid peroxidase and lactoperoxidase since the activity of horseradish peroxidase, myeloperoxidase, chloroperoxidase, or the pseudoperoxidase, metmyoglobin, was unaffected by incubation with resorcinol. The enzymatic oxidation of resorcinol by lactoperoxidase was linked to inactivation since the same products were observed spectrally, albeit at a much lower level, as were observed with horseradish peroxidase. The results are consistent with thyroid peroxidase- and lactoperoxidase-catalyzed oxidation of resorcinol derivatives to reactive radical species that covalently bind to amino acid residues unique to these two enzymes. The oxidation of thyroid peroxidase and lactoperoxidase by hydrogen peroxide produces catalytic intermediates containing unpaired electron density on amino acid residues similar to that seen with cytochrome c peroxidase. These results provide an explanation for the potency of resorcinol derivatives in the inhibition of LPO and TPO and the goitrogenic responses observed in humans and animals. The widespread occurrence of resorcinol derivatives in the environment suggests that exposure to these compounds may cause thyroid dysfunction in humans.

Dose-Response Relationship, Drug↗

PubMed1993

Chemical and enzymatic oxidation of benzimidazoline-2-thiones: a dichotomy in the mechanism of peroxidase inhibition.

Derivatives of imidazole-2-thiones block reactions catalyzed by thyroid peroxidase (TPX) and the closely related lactoperoxidase (LPX), and this property is used therapeutically to treat hyperthyroidism. The reactions of a series of benzimidazoline-2-thiones with chemical and enzymatic oxidants were investigated to probe systematically the mechanism of inhibition. Oxidation of benzimidazoline-2-thione (I) and 1-methylbenzimidazoline-2-thione (II) with 3-chloroperbenzoic acid (PBA) yielded reaction products and stoichiometry consistent with benzimidazole-2-sulfenic acids as reactive intermediates. The N,N'-disubstituted nature of 1,3-dimethylbenzimidazoline-2-thione (III) precludes sulfenic acid formation by tautomerization, and the oxidation of III with PBA yielded products and stoichiometry that were consistent with a benzimidazole-2-sulfonyl ylide as the reactive intermediate. I and II are suicide inhibitors of LPX and TPX, but III was found to inhibit only peroxidase-catalyzed iodination reactions by an alternate substrate mechanism. These results provide support for the hypothesis that imidazole-2-sulfenic acids are important reactive intermediates in the suicide inactivation of TPX and LPX and relate the chemical reactivity of the inhibitor with both the potency and mechanism of inhibition. These results suggest that 1,3-disubstituted thiourea derivatives represent a new class of potential antihyperthyroid drugs that block TPX-catalyzed tyrosine iodination but do not cause irreversible enzyme inactivation.

Benzimidazoles↗

PubMed1993

Analysis of clenbuterol in human plasma using liquid chromatography/atmospheric-pressure chemical-ionization mass spectrometry.

Clenbuterol is a beta-agonist drug used illegally as a growth stimulant in meat-producing animals and human athletes. The analysis of clenbuterol in spiked human plasma was performed using on-line liquid chromatography/atmospheric-pressure chemical-ionization mass spectrometry (LC/APCI-MS) using a conventional bore LC column (flow rate = 1.0 mL/min). At low sampling cone voltages, the mass spectrum was predominantly the [M+H]+ ion but diagnostic fragment ions were formed upon incremental increases in sampling cone voltage. The detection limit (signal-to-noise ratio of 3) using selected-ion monitoring of the [M+H]+ ion was 0.1 ng on-column (10 ppb). This is a 50-fold better sensitivity of detection than that previously reported for an on-line thermospray LC/MS method. The extraction procedures were not optimized for maximum sensitivity and the lack of interferences suggests that much lower detection limits for clenbuterol in plasma are attainable.

Clenbuterol↗

PubMed1992

Covalent binding of 14C- and 35S-labeled thiocarbamides in rat hepatic microsomes.

The covalent binding of a series of 14C- or 35S-labeled benzimidazole-2-thione (MBI) derivatives to rat liver microsomal proteins was studied to determine the mechanisms of hepatic monooxygenase oxidation of model anti-hyperthyroid compounds. All thiocarbamides tested (including methimazole) produced an NADPH-dependent loss of cytochrome P450 (P450) chromophore which could be prevented by the addition of glutathione (GSH). The covalent binding of MBI to liver microsomal proteins from dexamethasone (DEX)-pretreated rats was enhanced 10-fold with NADPH, unaffected by P450 inactivation with 1-aminobenzotriazole (ABT) and attenuated by GSH addition. Heat treatment of microsomes to inactivate the flavin-containing monooxygenase (FMO) decreased the observed binding. Equivalent amounts of [35S]- and [14C]MBI were covalently bound to hepatic microsomal proteins, suggesting retention of both the carbon and sulfur portions of the molecule in the MBI/protein adduct. Thiophilic reagents effected release of covalently bound [14C]- and [35S]MBI in equal amounts suggesting the presence of disulfide bonds between an MBI-derived sulfenic acid and microsomal protein thiols. Coincubation with bovine serum albumin (BSA) resulted in NADPH-dependent binding of [14C]-MBI to BSA sulfhydryls which was blocked by prior treatment of BSA with iodoacetamide. 1-Methyl-benzimidazole-2-thione (MMBI) also covalently bound to microsomal proteins and BSA but at levels lower than with MBI. P450, however, appeared to be more important than FMO in the metabolism of MMBI based on the effects of microsome heat pretreatment or ABT addition. In addition, ca. 1.5-fold more 35S- than 14C-label became bound. The covalent binding of [35S]1,3-dimethyl-benzimidazole-2-thione (DMMBI) to microsomal proteins was ca. six times greater than that of [14C]DMMBI. ABT, catalase and superoxide dismutase had a minimal effect on [35S]DMMBI binding, while FMO inactivation decreased binding by ca. 30%. These findings suggest that both monooxygenases contribute significantly to the hepatic metabolism of thiocarbamides. However, FMO activates thiocarbamides primarily to sulfenic acids, whereas P450 appears to produce both sulfenic acid and other reactive sulfur-derived metabolites. Thiol groups of P450 and other proteins are the molecular targets for these reactive species formed during the hepatic metabolism of anti-hyperthyroid drugs.

Animals↗

PubMed1991

Liquid chromatographic determination of ethylenethiourea using pulsed amperometric detection.

A liquid chromatographic method was developed using pulsed amperometric detection at a gold working electrode to measure residue levels of ethylenethiourea (ETU) in crops and groundwater. Use of the sequential pulsing program eliminates electrode fouling while preserving the sensitive and selective detection of ETU. Minimum detection limits in crops were 5-10 ppb (1.25-2.5 ng on-column) and 5 ppb (0.5 ng) in groundwater. The commercial availability of the pulsed electrochemical detector and its gold working electrode that remains functional with a minimum of conditioning is an improvement in method simplicity.

Chromatography, Liquid↗

PubMed1991

Peroxidase-catalyzed S-oxygenation: mechanism of oxygen transfer for lactoperoxidase.

The mechanism of organosulfur oxygenation by peroxidases [lactoperoxidase (LPX), chloroperoxidase, thyroid peroxidase, and horseradish peroxidase] and hydrogen peroxide was investigated by use of para-substituted thiobenzamides and thioanisoles. The rate constants for thiobenzamide oxygenation by LPX/H2O2 were found to correlate with calculated vertical ionization potentials, suggesting rate-limiting single-electron transfer between LPX compound I and the organosulfur substrate. The incorporation of oxygen from 18O-labeled hydrogen peroxide, water, and molecular oxygen into sulfoxides during peroxidase-catalyzed S-oxygenation reactions was determined by LC- and GC-MS. All peroxidases tested catalyzed essentially quantitative oxygen transfer from 18O-labeled hydrogen peroxide into thiobenzamide S-oxide, suggesting that oxygen rebound from the oxoferryl heme is tightly coupled with the initial electron transfer in the active site. Experiments using H2(18)O2, 18O2, and H2(18)O showed that LPX catalyzed approximately 85, 22, and 0% 18O-incorporation into thioanisole sulfoxide oxygen, respectively. These results are consistent with a active site controlled mechanism in which the protein radical form of LPX compound I is an intermediate in LPX-mediated sulfoxidation reactions.

Catalysis↗