Elsevier

Biochemical Pharmacology

Volume 44, Issue 10, 17 November 1992, Pages 2029-2037
Biochemical Pharmacology

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

https://doi.org/10.1016/0006-2952(92)90106-SGet rights and content

Abstract

The oxidation of thiourea, phenylthiourea, 1,3-diphenylthiourea, 1,3-bis-(3,4-dichloro-phenyl)-2-thiourea and 1,1-dibenzyl-3-phenyl-2-thiourea was measured in reactions catalyzed by purified pig liver flavin-containing monooxygenase (FMO-1) and by microsomal fractions isolated from pig, guinea pig, chicken, rat and rabbit tissues. The reactions, followed by measuring substrate-dependent thiocholine oxidation [Guo and Ziegler, Anal Biochem198: 143–148, 1991], were carried out in the presence of 2 mM 1-benzylimidazole to minimize potential interference from reactions other than those catalyzed by isoforms of the flavin-containing monooxygenase (FMO). While at saturating substrate concentrations the Vmax for purified FMO-1 catalyzed oxidation of all five thiocarbamides was essentially constant, velocities for the microsomal catalyzed reactions varied not only with tissue and species but also with the van der Waals' surface area of the thiocarbamide. Rat liver, rat kidney and rabbit liver microsomes failed to catalyze detectable oxidation of thiocarbamides larger than 1,3-diphenylthiourea and lung microsomes from a female rabbit only accepted substrates smaller than 1,3-diphenylthiourea. On the other hand, liver microsomes from chickens, pigs and guinea pigs catalyzed the oxidation of larger thiocarbamides, but the rates decreased with increasing substrate size and chicken liver microsomes showed no detectable activity with the largest thiocarbamide tested. To define more precisely the parameters affecting thiocarbamide substrate specificity of microsomal preparations, activities present in detergent extracts of guinea pig liver microsomes were separated into three distinct fractions. The substrate specificities of these partially purified fractions were different and consistent with the difference observed with microsomal catalyzed reactions. This strongly suggests that thiocarbamides that differ in size may be useful probes for measuring the number of activities of FMO isoforms in crude tissue preparations.

Cited by (37)

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

    2008, Biochemical Pharmacology
    Citation Excerpt :

    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 [2–5]. Humans have five FMO genes (FMO1-5) and six pseudogenes (FMO6P-11P) [6–8], while mice have nine FMO genes (Fmo1-6, 9, 12 and 13) [7].

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

    2005, Pharmacology and Therapeutics
    Citation Excerpt :

    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).

  • Thiourea toxicity in mouse C3H/10T1/2 cells expressing human flavin-dependent monooxygenase 3

    2002, Biochemical Pharmacology
    Citation Excerpt :

    The thiocarbamate specificity of FMO was originally delineated by the ability of a spectrum of these compounds to support NADPH oxidation in the presence of purified FMO 1 [22]. In this and subsequent studies, it was shown that the kinetic characteristics for TU (Vmax=1180 nmol/min/mg, Km=4 μM) were similar to ETU (Vmax=1060 nmol/min/mg, Km=99 μM), and PTU and ANTU [15,26]. When ETU was tested for cytotoxicity on parental and FMO 3 clone 7, it proved negative over a concentration range of 10−6 to 10−3 M.

View all citing articles on Scopus
View full text