Mechanism-Based Inactivation of Lactoperoxidase and Thyroid Peroxidase by Resorcinol Derivatives

Abstract

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.

Simple and rapid in vitro assay for detecting human thyroid peroxidase disruption

“…r 2 is indicated for the goodness-of-fit of a logistic dose-response model. The results obtained with hTPO also mirror the effect of the TPO inhibitors tested in humans (Divi and Doerge, 1994;Nakamura et al, 2007). hTPO inhibition profiles derived from both the luminol and Ampliflu Red assay showed that MMI followed by quercetin and resorcinol were the most potent of the inhibitors tested.…”

“…The association between the topical application of resorcinol in ointments and thyroid dysfunction in humans has a long history but even though rodent studies support causality, the higher sensitivity of rodents to thyroid disruption means that causality in humans is still considered inconclusive (Lynch et al, 2002). The resorcinol derivative quercetin, which is present in plant components of the human diet, is thought to be offsetting the benefit of iodized salt in parts of India that are affected by endemic goitre and while further human studies are necessary to confirm causality, recent rodent studies support the notion that quercetin inhibits thyroid function (Divi and Doerge, 1994;Giuliani et al, 2014). Overall, these antioxidants inhibit TPO in vitro and are associated with thyroid dysfunction in vivo, and this is accurately reflected in the luminol assay.…”

Resveratrol is a Peroxidase-mediated Inactivator of COX-1 but Not COX-2

“…We conclude that the mechanism-based inactivation of COX-1 is not accompanied by stable covalent modification of the enzyme. During the mechanism-based inactivation of lactoperoxidase (LPO) by resorcinol (m-hydroquinone), a stoichiometry of 10.0 mol of resorcinol incorporated/monomer was reported (27). However, SDS-PAGE and RP-HPLC were not used to determine whether stable covalent modification had occurred.…”

“…Both enzymes are likely to oxidize the m-hydroquinone to an unstabilized radical species, yet only in COX-1 does enzyme inactivation occur. Such an unstabilized radical was proposed for the inactivation of LPO and thyroid peroxidase (TPO) by resorcinol (27). Because stable covalent modification of COX-1 was not observed, inactivation must result from a "hit-and-run" mechanism in which the unstabilized m-hydroquinone radical generates a protein radical that goes on to inactivate the enzyme.…”

Goitrogenic and estrogenic activity of soy isoflavones.

“…The first step is iodination of thyroglobulin tyrosyl residues, followed by oxidative coupling to yield T 4 and T 3 . Inhibition of porcine TPO activity is a mechanism common to many classes of synthetic antithyroid compounds (27)(28)(29)(30)(31) and naturally occurring flavonoids (32,33). Lactoperoxidase (LPO) is often used as a model for TPO, based on many shared structural and functional properties.…”