Myeloperoxidase catalysed cooxidative metabolism of methimazole: Oxidation of glutathione and NADH by free radical intermediates

doi:10.1016/0009-2797(90)90009-C

Chemico-Biological Interactions

Volume 73, Issues 2–3, 1990, Pages 279-295

https://doi.org/10.1016/0009-2797(90)90009-C Get rights and content

Abstract

The myeloperoxidase catalysed oxidation of methimazole in the presence of NADH or GSH resulted in oxygen uptake suggesting that metabolism proceeded via a one electron mechanism. The GSH was oxidised to GSSG and the thiyl radical could be trapped with DMPO while NADH was oxidized to NAD⁺. Metabolism proceeded without the inactivation of the enzyme myeloperoxidase. Myeloperoxidase catalyzed oxidation of other substrates which proceed via one electron intermediates; 2,6-dimethylphenol, N,N,N′,N′-tetramethylphenylenediamine and luminol, were all stimulated by methimazole providing further evidence for a methimazole free radical. The presence of iodide stimulated the oxidation of methimazole but inhibited the oxygen uptake in the presence of GSH or NADH suggesting that metabolism in this case proceeded by a two electron mechanism. In contrast, another S-thioureylene drug, thiourea; did not cause oxygen uptake when oxidised in the presence of GSH or NADH indicating that the myeloperoxidase oxidation of thiourea proceeded primarily by a two electron mechanism. The horseradish peroxidase catalysed one electron oxidation of p′p′-biphenol, and 3,3′,5,5′-tetramethylbenzidine was reversibly inhibited by methimazole and thiourea by preventing the accumulation of oxidation products via reductive mechanisms whereas the reversible inhibition of guaiacol and luminol oxidation was the result of competitive inhibition. With p,p′-biphenol, and 3,3′,5,5′-tetramethylbenzidine unstable adduct formation could be demonstrated.

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Lactoperoxidase was previously used as a model enzyme to test the inhibitory activity of selenium analogs of anti-thyroid drugs with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. Peroxidases oxidize ABTS to a metastable radical ABTS^•+, which is readily reduced by many antioxidants, including thiol-containing compounds, and it has been used for decades to measure antioxidant activity in biological samples. We showed that anti-thyroid drugs 6-n-propyl-2-thiouracil, methimazole, and selenium analogs of methimazole also reduced it rapidly. This reaction may explain the anti-thyroid action of many other compounds, particularly natural antioxidants, which may reduce the oxidized form of iodine and/or tyrosyl radicals generated by thyroid peroxidase thus decreasing the production of thyroid hormones. However, influence of selenium analogs of methimazole on the rate of hydrogen peroxide consumption during oxidation of ABTS by lactoperoxidase was moderate. Direct hydrogen peroxide reduction, proposed before as their mechanism of action, cannot therefore account for the observed inhibitory effects. 1-Methylimidazole-2-selone and its diselenide were oxidized by ABTS^•+ to relatively stable seleninic acid, which decomposed slowly to selenite and 1-methylimidazole. In contrast, oxidation of 1,3-dimethylimidazole-2-selone gave selenite and 1,3-dimethylimidazolium cation. Accumulation of the corresponding seleninic acid was not observed.
Thyroid organotypic rat and human cultures used to investigate drug effects on thyroid function, hormone synthesis and release pathways
2012, Toxicology and Applied Pharmacology
Citation Excerpt :
MMI and PTU are typically used to treat hyperthyroidism, including Graves’ disease, resulting in returning thyroid function to within normal range (Abuid and Larsen, 1974; Nakamura et al., 2007; Roy and Mugesh, 2005; http://www.medicinenet.com/ methimazole/article.htm). Inhibition of TPO by MMI and PTU is mainly via the interaction of the thione group of these compounds with the metal center of TPO, comprising an iron-sulfur coordination which is common with metalloenzymes (McGirr et al., 1990; O'Brien, 2000; Tafazoli and O'Brien, 2005). These drugs may also trap iodine through the formation of stable donor-acceptor electron complexes with diiodine, depleting iodine for the iodination of thyroglobulin (Tg) (Brock and Head, 1966; Marchant et al., 1971, 1972; Raby et al., 1990; Roy and Mugesh, 2006; Wartofsky and Ingbar, 1971).
Drug induced thyroid effects were evaluated in organotypic models utilizing either a rat thyroid lobe or human thyroid slices to compare rodent and human response. An inhibition of thyroid peroxidase (TPO) function led to a perturbation in the expression of key genes in thyroid hormone synthesis and release pathways. The clinically used thiourea drugs, methimazole (MMI) and 6-n-propyl-2-thioruacil (PTU), were used to evaluate thyroid drug response in these models. Inhibition of TPO occurred early as shown in rat thyroid lobes (2 h) and was sustained in both rat (24–48 h) and human (24 h) with ≥ 10 μM MMI. Thyroid from rats treated with single doses of MMI (30–1000 mg/kg) exhibited sustained TPO inhibition at 48 h. The MMI in vivo thyroid concentrations were comparable to the culture concentrations (~ 15–84 μM), thus demonstrating a close correlation between in vivo and ex vivo thyroid effects. A compensatory response to TPO inhibition was demonstrated in the rat thyroid lobe with significant up-regulation of genes involved in the pathway of thyroid hormone synthesis (Tpo, Dio1, Slc5a5, Tg, Tshr) and the megalin release pathway (Lrp2) by 24 h with MMI (≥ 10 μM) and PTU (100 μM). Similarly, thyroid from the rat in vivo study exhibited an up-regulation of Dio1, Slc5a5, Lrp2, and Tshr. In human thyroid slices, there were few gene expression changes (Slc5a5, ~ 2-fold) and only at higher MMI concentrations (≥ 1500 μM, 24 h). Extended exposure (48 h) resulted in up-regulation of Tpo, Dio1 and Lrp2, along with Slc5a5 and Tshr. In summary, TPO was inhibited by similar MMI concentrations in rat and human tissue, however an increased sensitivity to drug treatment in rat is indicated by the up-regulation of thyroid hormone synthesis and release gene pathways at concentrations found not to affect human tissue.
Blood cell oxidative stress precedes hemolysis in whole blood-liver slice co-cultures of rat, dog, and human tissues
2010, Toxicology and Applied Pharmacology
A novel in vitro model to investigate time-dependent and concentration-dependent responses in blood cells and hemolytic events is studied for rat, dog, and human tissues. Whole blood is co-cultured with a precision-cut liver slice. Methimazole (MMI) was selected as a reference compound, since metabolism of its imidazole thione moiety is linked with hematologic disorders and hepatotoxicity. An oxidative stress response occurred in all three species, marked by a decline in blood GSH levels by 24 h that progressed, and preceded hemolysis, which occurred at high MMI concentrations in the presence of a liver slice with rat (≥ 1000 μM at 48 h) and human tissues (≥ 1000 μM at 48 h, ≥ 750 μM at 72 h) but not dog. Human blood-only cultures exhibited a decline of GSH levels but minimal to no hemolysis. The up-regulation of liver genes for heme degradation (Hmox1 and Prdx1), iron cellular transport (Slc40a1), and GSH synthesis and utilization (mGST1 and Gclc) were early markers of the oxidative stress response. The up-regulation of the Kupffer cell lectin Lgals3 gene expression indicated a response to damaged red blood cells, and Hp (haptoglobin) up-regulation is indicative of increased hemoglobin uptake. Up-regulation of liver IL-6 and IL-8 gene expression suggested an activation of an inflammatory response by liver endothelial cells. In summary, MMI exposure led to an oxidative stress response in blood cells, and an up-regulation of liver genes involved with oxidative stress and heme homeostasis, which was clearly separate and preceded frank hemolysis.
Glutathione-induced radical formation on lactoperoxidase does not correlate with the enzyme's peroxidase activity
2007, Free Radical Biology and Medicine
Lactoperoxidase (LPO) is believed to serve as a mediator of host defense against invading pathogens. The protein is more abundant in body fluids such as milk, saliva, and tears. Lactoperoxidase is known to mediate the oxidation of halides and (pseudo)halides in the presence of hydrogen peroxide to reactive intermediates presumably involved in pathogen killing. More recently, LPO has been shown to oxidize a wide diversity of thiol compounds to thiyl free radicals, which ultimately lead to the formation of a protein radical characterized by DMPO-immunospin trapping. In the same study by our group the authors claimed that a consequence of this protein radical formation was the inactivation of LPO (Guo et al., J. Biol. Chem. 279:13272–13283; 2004). Here we demonstrate that although thiyl radical formation does lead to LPO radical production, the formation of this radical is unrelated to the enzyme's activity. We suggest the source of this misleading interpretation to be the binding of GSH to ELISA plates, which interferes with ABTS and guaiacol oxidation. In addition, DMPO-GSH-nitrone adducts bind to ELISA plates, leading to ambiguities of interpretation since we have demonstrated that DMPO-GSH nitrone does not bind to LPO, and only LPO-protein-DMPO-nitrone adducts can be detected by Western blot.
Peroxidase catalysed formation of cytotoxic prooxidant phenothiazine free radicals at physiological pH
2004, Chemico-Biological Interactions
Citation Excerpt :
The biological activity of these drugs depends on the physicochemical properties of the drug, the type of target tissue acted on, and the presence of functional groups on the phenothiazine lead structure [4]. Previously, we showed that the sulfur radical of methimazole (methimazole thiyl radical) formed by myeloperoxidase or horseradish peroxidase co-oxidised glutathione and NADH to form reactive oxygen species [5]. The methimazole thiyl radical was implicated in the inactivation of thyroid peroxidase, possibly explaining the therapeutic usefulness of this compound for treating hyperthyroidism [6].
The antipsychotic phenothiazines may have other therapeutic applications because of their ability to kill bacteria, plasmids and tumor cells. They are also known to undergo a peroxidase-catalysed oxidation to form cation radicals that are stable at acid pH, but are not detected at a neutral pH. The objective of this project was to determine whether phenothiazine cation radical metabolites could cause oxidative stress at a neutral pH resulting in cytotoxicity. At a neutral pH, catalytic amounts of phenothiazines were found to be oxidised by a peroxidase/H₂O₂ system and also caused ascorbate, GSH and NADH cooxidation. NADH and GSH co-oxidation was accompanied by oxygen uptake and was increased by the addition of catalytic amounts of superoxide dismutase, indicating that the superoxide radical was formed. The phenothazines were different from other peroxidase substrates in that the NADH, ascorbate or GSH cooxidation was faster at pH 6.0 than pH 7.4, thereby partly reflecting the cation radical stability. The order of catalytic effectiveness found was promazine > chlorpromazine > trifluoperazine. Peroxidase/H₂O₂ also markedly increased phenothiazine cytotoxicity towards isolated rat hepatocytes at nontoxic phenothiazine concentrations. At both pH 6.0 and 7.4, the same order of phenothiazine catalytic effectiveness was observed as seen in the co-oxidation experiments. Cytotoxicity to hepatocytes could be attributed to oxidative stress as most hepatocyte glutathione oxidation and lipid peroxidation preceeded phenothiazine induced cytotoxicity and that cytotoxicity was prevented by the antioxidant butylated hydroxyanisole. This hepatocyte/peroxidase/H₂O₂ system could be a useful model for studying drug induced idiosyncratic hepatic injury enhanced by inflammation.
Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: Glutathione oxidation and conjugation
2001, Free Radical Biology and Medicine
GSH was readily depleted by a flavonoid, H₂O₂, and peroxidase mixture but the products formed were dependent on the redox potential of the flavonoid. Catalytic amounts of apigenin and naringenin but not kaempferol (flavonoids that contain a phenol B ring) when oxidized by H₂O₂ and peroxidase cooxidized GSH to GSSG via a thiyl radical which could be trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a DMPO-glutathionyl radical adduct detected by ESR spectroscopy. On the other hand, quercetin and luteolin (flavonoids that contain a catechol B ring) or kaempferol depleted GSH stoichiometrically without forming a thiyl radical or GSSG. Quercetin, luteolin, and kaempferol formed mono-GSH and bis-GSH conjugates, whereas apigenin and naringenin did not form GSH conjugates. MS/MS electrospray spectroscopy showed that mono-GSH conjugates for quercetin and luteolin had peaks at m/z 608 [M + H]⁺ and m/z 592 [M + H]⁺ in the positive-ion mode, respectively. ¹H NMR spectroscopy showed that the GSH was bound to the quercetin A ring. Spectral studies indicated that at a physiological pH the luteolin-SG conjugate was formed from a product with a UV maximum absorbance at 260 nm that was reducible by potassium borohydride. The quercetin-SG conjugate or kaempferol-SG conjugate on the other hand was formed from a product with a UV maximum absorbance at 335 nm that was not reducible by potassium borohydride. These results suggest that GSH was oxidized by apigenin/naringenin phenoxyl radicals, whereas GSH conjugate formation involved the o-quinone metabolite of luteolin or the quinoid (quinone methide) product of quercetin/kaempferol.

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Myeloperoxidase catalysed cooxidative metabolism of methimazole: Oxidation of glutathione and NADH by free radical intermediates

Abstract

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Chem.-Biol. Interact.

Chem.-Biol. Interact.

Free Rad. Biol. Med.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

FEBS Lett.

Antithyroid drugs

Pharmacol. Ther., B.

The thyroid accumulation, oxidation and metabolic fate of 35S-methimazole in the rat

Endocrinology

Mechanisms of action of thioureylene antithyroid drugs: factors affecting intrathyroidal metabolism of propylthiouracil and methimazole in rats

Endocrinology

Reversible and irreversible inhibition of thyroid peroxidase catalyzed iodination by thioureylene drugs

Endocrinology

The irreversible inactivation of thyroid peroxidase by methyl mercaptoimidazole, thiouracil and propylthiouracil, in vitro, and its relationship to in vivo findings

Endocrinology

Mechanism based inhibition of lactoperoxidase by thiocarbamide goitrogens

Biochemistry

The thyroid accumulation, oxidation and metabolic fate of ³⁵S-methimazole in the rat