Use of thiocarbamides as selective substrate probes for isoforms of flavin-containing monooxygenases

Guo, Wei-Xi A.; Poulsen, Lawrence L.; Ziegler, Daniel M.

doi:10.1016/0006-2952(92)90106-s

Cited by 33 publications

(42 citation statements)

References 18 publications

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“…We followed the reaction at pH 9.5 to compensate for the lower sensitivity provided by this general assay. Like previous studies performed with rFMO2 (Nagata et al, 1990;Guo et al, 1992), hFMO2.1 is active toward thiourea compounds with a small crosssectional area but is inactive toward 1,3-diphenylthiourea, which has a larger cross-section (11.2 Å). However, unlike rFMO2, which has a 10-fold increase in K m , the K m of hFMO2.1 decreased 6-fold when 1-phenylthiourea was the substrate rather than thiourea (2.4 Å).…”

Section: Discussionsupporting

confidence: 74%

Identification of Active Flavin-Containing Monooxygenase Isoform 2 in Human Lung and Characterization of Expressed Protein

Krueger

Martin²,

Yueh³

et al. 2002

Drug Metab Dispos

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This paper is available online at http://dmd.aspetjournals.org ABSTRACT:Full-length human (hFMO2.1) and monkey (mFMO2) flavin-containing monooxygenase proteins, which share 97% sequence identity, were produced by baculovirus-mediated expression in insect cells and assayed for S-oxygenation under conditions known to affect FMO activity. Both enzymes demonstrated maximal activity at pH 9.5; but hFMO2.1 retained significantly more activity than mFMO2 did at pH 9.0 and higher. hFMO2.1 also retained significantly more activity than mFMO2 did in the presence of magnesium and all detergents tested. Although hFMO2.1 had more residual activity after heating at 45°C than mFMO2, under some conditions, both had less than 10% of control activity, whereas expressed rabbit FMO2 retained over 50% activity. Screening for NADPH-oxygenation by hFMO2.1, indicated that substituted thioureas with a small cross-sectional area (2.4-4.3 Å) are good substrates, whereas 1,3-diphenylthiourea (11.2 Å) was not oxygenated. We confirmed the presence of hFMO2.1 in lung tissue from a heterozygous individual (hFMO2*1/hFMO2*2A) by Western analysis and confirmed activity by S-oxygenation. These microsomes also demonstrated a heatassociated loss of activity similar to expressed hFMO2.1. The heat sensitivity of hFMO2.1 may partially explain why activity in post mortem human lung samples has previously been unreported. Individuals that have the FMO2*1 allele-encoding full-length hFMO2.1 may exhibit altered drug metabolism in the lung.The mammalian flavin-containing monooxygenases (FMO 3 ; EC 1.14.13.8) are a family (each family having a single member) of xenobiotic-metabolizing enzymes that bind FAD as a prosthetic group and NADPH as a cofactor. Substrates are structurally diverse compounds containing a soft nucleophile; although this is commonly nitrogen or sulfur (Ziegler, 1993;Cashman, 1995;Cashman et al., 2000), other nucleophiles, such as some selenium-containing compounds, are also substrates (Chen and Ziegler, 1994). Metabolism by FMO generally yields metabolic products that are more polar and less toxic or less biologically active than the parent xenobiotic, as is the case for tertiary amines (Ziegler, 1984;Damani, 1988). However, bioactivation, often involving sulfur oxygenation, sometimes results (Ziegler, 1991;Cashman, 1995;Genter et al., 1995).Proteins from four forms of FMO have been confirmed (FMOs 1-3 and 5) by immunodetection in human tissue samples (Haining et al., 1997;Myers et al., 1997;Overby et al., 1997; Whetstine et al., 2000;Yeung et al., 2000). Expression of protein from FMO4 and a recently identified sixth isoform on human chromosome 1 (ENTREZ accession AL021026) has not been demonstrated yet. FMO isoforms can be distinguished on the basis of patterns of tissue and developmental expression as exemplified by human isoforms 1 and 3 (Dolphin et al., 1996). In addition, there are isoform differences in substrate specificity determined, in part, by the size and shape of the nucleophilic xenobiotic and isoform-dependent stereosel...

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Section: Discussionsupporting

confidence: 74%

Identification of Active Flavin-Containing Monooxygenase Isoform 2 in Human Lung and Characterization of Expressed Protein

Krueger

Martin²,

Yueh³

et al. 2002

Drug Metab Dispos

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“…The lack of activity of rabbit FMO2 toward imipramine and chlorpromazine are consistent with this model. Thioureas of various sizes have been used to demonstrate the existence of multiple FMO enzymes in various tissues (Nagata et al, 1990;Guo et al, 1992;Shehin-Johnson et al, 1995;Kim & Ziegler, 2000). The activity of rabbit FMO2 toward alkyl 2-naphthyl sulfides of increasing chain length confirm this restricted substrate access channel in FMO2 (Fisher & Rettie, 1997).…”

Section: Substrate Specificity Of Enzyme-restricted Substrate Access mentioning

confidence: 99%

“…FMOs bioactivate thioureas through S-oxygenation to toxic sulfenic and/or sulfinic acid metabolites Neal & Halpert, 1982;Ziegler, 1982;Sabourin & Hodgson, 1984;Nagata et al, 1990;Decker & Doerge, 1991;Guo et al, 1992;Kim & Ziegler, 2000;Smith & Crespi, 2002). The sulfenic acid is capable of undergoing redox cycling following conjugation with glutathione, resulting in oxidative stress due to depletion of reduced glutathione and NADPH (Hardwick et al, 1991;Onderwater et al, 1998Onderwater et al, , 1999Onderwater et al, , 2004Smith & Crespi, 2002;Henderson et al, 2004b).…”

Section: Genetic Polymorphisms Of Flavin-containing Monooxygenasementioning

confidence: 99%

“…FMO can S-oxygenate thioureas to sulfenic acids which can undergo redox cycling or be further converted to the reactive sulfinic acid Neal & Halpert, 1982;Ziegler, 1982;Krieter et al, 1984;Sabourin & Hodgson, 1984;Nagata et al, 1990;Decker & Doerge, 1991;Guo & Ziegler, 1991;Guo et al, 1992;Onderwater et al, 1999Onderwater et al, , 2004Kim & Ziegler, 2000;Smith & Crespi, 2002;Henderson et al, 2004b).…”

Section: Introductionmentioning

confidence: 99%

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Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism

Krueger

Williams

2005

Pharmacology & Therapeutics

493

444

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Flavin-containing monooxygenase (FMO) oxygenates drugs and xenobiotics containing a "softnucleophile", usually nitrogen or sulfur. FMO, like cytochrome P450 (CYP), is a monooxygenase, utilizing the reducing equivalents of NADPH to reduce 1 atom of molecular oxygen to water, while the other atom is used to oxidize the substrate. FMO and CYP also exhibit similar tissue and cellular location, molecular weight, substrate specificity, and exist as multiple enzymes under developmental control. The human FMO functional gene family is much smaller (5 families each with a single member) than CYP. FMO does not require a reductase to transfer electrons from NADPH and the catalytic cycle of the 2 monooxygenases is strikingly different. Another distinction is the lack of induction of FMOs by xenobiotics.In general, CYP is the major contributor to oxidative xenobiotic metabolism. However, FMO activity may be of significance in a number of cases and should not be overlooked. FMO and CYP have overlapping substrate specificities, but often yield distinct metabolites with potentially significant toxicological/pharmacological consequences.The physiological function(s) of FMO are poorly understood. Three of the 5 expressed human FMO genes, FMO1, FMO2 and FMO3, exhibit genetic polymorphisms. The most studied of these is FMO3 (adult human liver) in which mutant alleles contribute to the disease known as trimethylaminuria. The consequences of these FMO genetic polymorphisms in drug metabolism and human health are areas of research requiring further exploration.

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“…Although oxygenation usually represents a detoxication reaction, some FMO-oxygenated sulfur substrates, such as thioureas, produce reactive sulfenic or sulfinic acids as major metabolites. Sulfenic acid metabolites can react with GSH inducing oxidative stress through a futile redox cycle [2][3][4][5].…”

Section: Introductionmentioning

confidence: 99%

Characterization of mouse flavin-containing monooxygenase transcript levels in lung and liver, and activity of expressed isoforms

Siddens

Henderson

VanDyke

et al. 2008

Biochemical Pharmacology

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The significance of active vs. inactive flavin-containing monooxygenase 2 (FMO2) for human drug and xenobiotic metabolism and sensitivity is unknown, but the underlying ethnic polymorphism is well documented. We used quantitative real-time PCR to measure message levels of Fmo1, Fmo2, Fmo3 and Fmo5 in lung and liver from eight strains of eight week old female mice to determine if a strain could be identified that predominately expressed Fmo2 in lung, recapitulating the human FMO expression profile and being the ideal strain for Fmo2 knockout studies. We also characterized enzyme activity of baculovirus expressed mouse Fmo1, Fmo2 and Fmo3 to identify a substrate or incubation conditions capable of discriminating Fmo2 from Fmo mixtures. Fmo transcript expression patterns were similar for all strains. In lung, 59% of total FMO message was Fmo2, but Fmo1 levels were also high, averaging 34%, whereas Fmo3 and Fmo5 levels were 2 and 5%, respectively. In liver, Fmo1, Fmo2, Fmo3 and Fmo5 contributed 16, 1, 7 and 76% respectively, of detected message. Peak activity varied by isoform and was pH-and substrate-dependent. Fmo3 oxidation of methyl ptolyl sulfide was negligible at pH 9.5, but Fmo3 oxidation of methimazole was comparable to Fmo1 and Fmo2. Heating microsomes at 50 °C for 10 min eliminated most Fmo1 and Fmo3 activity, while 94% of Fmo2 activity remained. Measurement of activity in heated and unheated lung and liver microsomes verified relative transcript abundance. Our results show that dual Fmo1/2 knockouts will be required to model the human lung FMO profile.

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Use of thiocarbamides as selective substrate probes for isoforms of flavin-containing monooxygenases

Cited by 33 publications

References 18 publications

Identification of Active Flavin-Containing Monooxygenase Isoform 2 in Human Lung and Characterization of Expressed Protein

Identification of Active Flavin-Containing Monooxygenase Isoform 2 in Human Lung and Characterization of Expressed Protein

Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism

Characterization of mouse flavin-containing monooxygenase transcript levels in lung and liver, and activity of expressed isoforms

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