Evaluation of Xenobiotic N- and S-Oxidation by Variant Flavin-Containing Monooxygenase 1 (FMO1) Enzymes

Bjarte Furnes1 and Daniel Schlenk

Environmental Toxicology Program, University of California, Riverside, California 92521

Received November 25, 2003; accepted January 20, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The flavin-containing monooxygenase gene family (FMO16) in humans encodes five functional isoforms that catalyze the monooxygenation of numerous N-, P- and S-containing drugs and toxicants. A previous single nucleotide polymorphism (SNP) analysis of FMO1 in African-Americans identified seven novel SNPs. To determine the functional relevance of the coding FMO1 variants (H97Q, I303V, I303T, R502X), they were heterologously expressed using a baculovirus system. Catalytic efficiency and stereoselectivity of N- and S-oxygenation was determined in the FMO1 variants using several substrates. The I303V variant showed catalytic constants equal to wild-type FMO1 for methimazole and methyl p-tolyl sulfide. Catalytic efficiency (Vmax/Km) of methyl p-tolyl sulfide oxidation by R502X was unaltered. In contrast, methimazole oxidation by R502X was not detected. Both H97Q and I303T had elevated catalytic efficiency with regards to methyl p-tolyl sulfide (162% and 212%, respectively), but slightly reduced efficiency with regards to methimazole (81% and 78%). All the variants demonstrated the same stereoselectivity for methyl p-tolyl sulfide oxidation as wild-type FMO1. FMO1 also metabolized the commonly used insecticide fenthion to its (+)-sulfoxide, with relatively high catalytic efficiency. FMO3 metabolized fenthion to its sulfoxide at a lower catalytic efficiency than FMO1 (27%) and with less stereoselectivity (74% (+)-sulfoxide). Racemic fenthion sulfoxide was a weaker inhibitor of acetylcholinesterase than its parent compound (IC50 0.26 and 0.015 mM, respectively). The (+)- and (-)-sulfoxides were equally potent inhibitors of acetylcholinesterase. These data indicate that all the currently known FMO1 variants are catalytically active, but alterations in kinetic parameters were observed.

Key Words: flavin-containing monooxygenase; fenthion; thiourea; methimazole; African-American; polymorphisms.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The flavin-containing monooxygenases (FMOs) are a family of nicotinamide adenine dinucleotide phosphate (NADPH)- and oxygen-dependent enzymes that readily N- and S-oxygenate a diverse group of xenobiotics and certain endogenous amines (Cashman, 1995Go; Ziegler, 1988Go). Each isoform demonstrates species- and tissue-specific expression patterns as well as distinguishable but overlapping substrate specificities with other FMOs and cytochrome P450s (P450). In humans, six genes (FMO1FMO6) have been identified, but only expression of the FMO1–3 and 5 proteins has been detected.

FMO1 is the major liver isoform in most mammals examined to date, except humans. In adult humans, FMO3 is the major liver isoform, while FMO1 is expressed primarily in the kidney (47 pmol/mg) and the fetal liver (14.4 pmol/mg) (Yeung et al., 2000Go). Lower levels of FMO1 also have been detected in the intestines. Considerable interindividual differences also are observed (Koukouritaki et al., 2002Go). Hepatic FMO expression undergoes significant changes during development: FMO1 expression is highest in the embryo at 8–15 weeks gestation, and suppression occurs within 3 days after birth (Koukouritaki et al., 2002Go). Onset of FMO3 expression is highly variable but is detectable in most individuals at the age of 1–2 years.

Molecular mechanisms explaining the regulation of FMO1 and FMO3 in humans and rabbits have been partially elucidated. Regulatory domains containing binding sites for Yin Yang 1 (YY1), HNF1{alpha}, and HNF4{alpha} have been identified in the major FMO1 human and rabbit promoter (Hines et al., 2003Go; Luo and Hines, 2001Go). The reason for the switch between FMO1 and FMO3 expression is not known. Unlike the cytochrome P450 family, the FMOs do not seem to be induced by chemicals or by diet, although, in some instances they seem to be under hormonal regulation (Miller et al., 1997Go). High levels of FMO1 expression (combined with the high catalytic activity) suggest that it might be important in the metabolic clearance of sulfides and tertiary amines in humans at specific stages of development.

Sequence polymorphisms in FMO3 have been described in detail (Cashman and Zhang, 2002Go). Functional characterization of these variants has been helpful in linking deficiency in FMO3 catalytic activity to the inborn disease trimethylaminuria (Treacy et al., 1998Go). Trimethylaminuria is caused by a reduced N-oxygenation of trimethylamine, leading to the excretion of the odorous compound through exhaled air, sweat, and urine. Genetic characterization of FMO1 has only recently been initiated, but to our knowledge, no work has yet been published on functional studies of single nucleotide polymorphisms (SNPs) found in the coding regions of FMO1. We recently identified seven SNPs in FMO1 in African-Americans: H97Q (FMO1*2), I303V (FMO1*3), I303T (FMO1*4), R502X (FMO1*5), T249T (FMO1*1B), V396V (FMO1*1C), and an additional intronic variant, IVS3-11 T>C (Furnes et al., 2003Go). Two of these variants are in highly conserved amino acids, I303T and R502X (Table 1). The variants identified all had allelic frequencies of 2% or less. None of the individuals were homozygous for the FMO1 variants.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Amino Acid Alignments of the FMO1 H97, I303, and R502 Residues

 
Early studies examining the role of FMOs in xenobiotic biotransformation indicated significant activities toward sulfur-containing pesticides (Hajjar and Hodgson, 1980Go). Previous studies in our laboratory indicated FMO1 was the predominant FMO isoform that catalyzed the sulfoxidation and subsequent bioactivation of the acetylcholinesterase inhibitor, aldicarb (Schlenk et al., 2002Go). Another acetylcholinesterase inhibitor that has been shown to be an FMO substrate is the organophosphate fenthion (Venkatesh et al., 1991Go). Fenthion differs from many well-known organophosphates in that the signs of toxicity develop slower but are more persistent (Dubois and Kinoshita, 1964Go; Francis and Barnes, 1963Go). The involvement of the FMO system in human fenthion metabolism has not yet been well characterized.

Given the important role of sulfoxidation in the biotransformation and toxicity of sulfur-containing xenobiotics, and the expression of FMO1 within the fetal liver and adult kidney, the purpose of this study was to evaluate the catalytic efficiency and stereoselectivity of FMO1 variants that were heterologously expressed in a baculovirus system. Our results indicate that all the currently known FMO1 alleles encode catalytically active proteins and that FMO1 metabolizes fenthion with higher efficiency than FMO3.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Standard chemicals used (NADPH, flavin-adenine dinucleotide [FAD], methimazole, phenylmethylsulfonylfluoride [PMSF]) were from Sigma-Aldrich (St. Louis, MO) or VWR (West Chester, PA) and were of the highest purity commercially available. Solvents used for HPLC were HPLC grade. Imipramine, methyl p-tolyl sulfide, and the methyl p-tolyl (R)- and (S)-sulfoxides were obtained from Sigma-Aldrich. Fenthion and fenthion sulfoxide were purchased from Chem Service (West Chester, PA).

Site-directed mutagenesis.
An FMO1 cDNA clone was obtained as a gift from Dr. Allan Rettie (Department of Medicinal Chemistry, University of Washington, Seattle). The FMO3 wild-type and common K158L variant cDNAs were provided by Dr. John Cashman (Human BioMolecular Research Institute, San Diego). Site-directed mutagenesis was carried out using the Stratagene Quikchange XL protocol. Oligonucleotide primers used in the mutagenesis are summarized in Table 2. Sequence changes were confirmed by ABI sequencing on both DNA strands. Following site-directed mutagenesis, the FMO cDNAs were ligated into the pFastbac1 vector.


View this table:
[in this window]
[in a new window]
 
TABLE 2 Oligonucleotides Used in FMO1 and FMO3 Site-Directed Mutagenesis

 
Production of recombinant bacmids.
Production of recombinant bacmids was achieved by transforming the DH10Bac bacterial strain with the pFastbac1-FMO constructs. The DH10Bac strain is capable of site-specific transposition of an expression cassette from pFastbac1 into the bacmid bMON14272. The bacmids were isolated by a modified alkaline-lysis method and subsequently confirmed using PCR with M13F (5'-CCCAGTCACGACGTTGTAAAA-3') and M13R (5'-AGCGGATAACAATTTCACACA-3') primers.

Production of viral stocks and recombinant protein.
Sf9 cells were maintained in Sf-900 II serum-free medium (Invitrogen, Carlsbad, CA) containing 50 units/ml penicillin and 50 µg/ml streptomycin at 27.5°C. Sf9 cells were seeded to a density of 9 x 105 cells per well in a 35-mm 6-well plate and transfected with the recombinant bacmids using Cellfectin® (Invitrogen). The virus supernatant was harvested after 72 h and used to infect a 50 ml culture of Sf9 at 106 cells/ml. The cells were harvested after 48 h and assayed for FMO1 or FMO3 expression. The virus supernatant was titered and used to infect a 600 ml (1.0–1.3 x 106 cells/ml) culture of Sf9 cells for production of recombinant protein. FAD was added to a final concentration of 10 µg/ml 12 h after infection. Optimal infection conditions for production of recombinant protein were a Multiplicity of Infection (MOI) of 8–9 followed by vigorous shaking for 70–75 h.

Microsome preparation.
Cells were harvested by centrifugation at 750 x g for 5 min and immediately washed in storage buffer (50 mM potassium phosphate, 1 mM ethylenediaminetetraacetic acid [EDTA], and 20% glycerol, pH 7.4). Cells were homogenized in a glass/Teflon homogenizer using a buffer containing 1.15 M KCl, 10 mM EDTA, 100 mM potassium phosphate, 0.2 mM PMSF, pH 7.5. After differential centrifugation (12,000 x g for 12 min; 100,000 x g for 90 min), the pellet was resuspended to a concentration of 2–4 mg/ml in storage buffer.

Immunoquantitation of FMO1 and FMO3.
FMO1 and FMO3 at known concentrations were obtained from Gentest (Woburn, MA) and used to quantify the expressed FMO isoforms. SDS-PAGE and immunoblot was carried out as previously described (Laemmli, 1970Go; Towbin et al., 1979Go). Since the R502X is a truncated protein, a 16% separation gel was used in order to separate the R502X variant from the other full-length FMO1 variants. Densitometry analysis and quantitation were done by Quantity One® (Bio-Rad, Hercules, CA). Determination of total microsomal protein content was carried out by the Bradford assay (Bradford, 1976Go). Microsome preparations were stored at –80°C and only allowed to thaw once for catalytic assays.

NADPH oxidation assay.
Imipramine, methimazole, and thiourea oxidation was measured by substrate-dependent NADPH oxidation (340 nm) at 37°C (Wyatt et al., 1998Go). The reaction mixture contained 75–100 µg microsomal protein in 50 mM phosphate buffer (pH 8.4) containing 0.2 mM NADPH. The sample was allowed to equilibrate for 3 min before adding substrate. Substrate concentrations of 2.5–250 µM were used in a total reaction volume of 1 ml. All incubations were carried out in triplicate. An NADPH extinction coefficient of 6220 M–1 cm–1 was used in calculating catalytic constants.

Methyl p-tolyl sulfide sulfoxidation assay.
Assay of methyl p-tolyl sulfide sulfoxidation was carried out using a modified protocol based on the Gentest FMO1 SupersomesTM data sheet. All incubations were carried out in triplicate. A 0.25-ml reaction volume containing 75–100 µg microsomal protein, 1.3 mM NADPH, 3.3 mM MgCl2, and 1–1000 µM methyl p-tolyl sulfide in a 50 mM glycine buffer (pH 9.0) was incubated at 37°C for 10 min. Heat lability of the FMO1 variants was investigated by preincubating the samples at 45°C for 10 min in the absence of NAPDH. The reaction was stopped by the addition of 75 µl acetonitrile and centrifuged for 5 min at 10,000 x g. The supernatant was filtered over a Millipore Durapore (Bedford, MA) membrane and then analyzed on a Regis Technologies (R, R) Whelk-01 10/100 chromasil chiral column. The methyl p-tolyl sulfoxide was eluted with an initial methanol concentration of 46% (v/v) (0–7 min) that was slowly increased to 100% (7–20 min). Pure (R)- and (S)-methyl p-tolyl sulfoxides from Sigma-Aldrich were used to establish a standard curve. The (R)- and (S)-enantiomers were eluted with retention times of 12.9 and 13.7 min, respectively.

Fenthion sulfoxidation assay.
Incubation of the FMO1 variants in the presence of fenthion were carried out as described for methyl p-tolyl sulfide. Thirteen different concentrations of fenthion ranging from 1 to 1500 µM were used. The fenthion sulfoxides were eluted with retention times of 22 min (peak 1) and 31 min (peak 2) using a mobile phase consisting of hexane:isopropanol:dichloromethane (7:3:1). Stereoselectivity of FMO3 was determined at a single fenthion concentration of 1000 µM, and the kinetic parameters were determined following analysis of fenthion sulfoxide on a J'sphereTM ODS-L80 column using a 35% acetonitrile/65% water mobile phase. Fenthion sulfoxide had a retention time of 12.1 min under these conditions.

Determination of optical activity of fenthion sulfoxides.
Peak 1 and peak 2 of the resulting chromatogram were collected and reanalyzed to determine purity (Fig. 1). A Jasco J-600 circular dichroism spectrometer was used to establish the optical activity of the fenthion sulfoxides. The fenthion sulfoxide enantiomers were designated as either (+) or (–) if the difference in absorbance between left-circularly and right-circularly (AL –AR) polarized light was positive or negative, respectively.



View larger version (9K):
[in this window]
[in a new window]
 
FIG. 1. HPLC chromatogram of separated fenthion sulfoxide stereoisomers. (A) A racemic mixture of fenthion sulfoxide was separated on a chiral HPLC column under the conditions described in Materials and Methods. The two fractions corresponding to peak 1 (20–25 min) and peak 2 (30–35 min) were collected from the column. (B) The fractions corresponding to the two peaks were reanalyzed to determine purity. The optical activity was later established by circular dichroism. Peak 1 and 2 were designated as (+) and (–), respectively, if the difference in absorbance between left-circularly and right-circularly polarized light was positive or negative.

 
Acetylcholinesterase inhibition assay.
Purified recombinant human acetylcholinesterase was obtained from Sigma-Aldrich. Acetylcholinesterase activity was assayed as described by Ellman and coworkers (1961), but modified to a microplate assay. Briefly, a200-µl reaction volume consisting of 0.1 M potassium phosphate buffer, pH 7.4, 0.3 mM 5,5'-dithiobis-2-nitrobenzoic acid and 0.003 U of acetylcholinesterase was preincubated with fenthion, racemic fenthion sulfoxide, or the (+)- and (–)- sulfoxide 30 min before adding acetylthiocholine iodide (1–1000 µM). An IC50 was determined at an acetylthiocholine concentration equal to Km and five concentrations of fenthion or fenthion sulfoxide. Microplates were read on a Molecular Devices (Sunnyvale, CA) microplate reader and analyzed using the software package SOFTmax® Pro.

Statistics.
Kinetic parameters were determined using Prism 3.0 (Graphpad Software, San Diego, CA). A one-site binding model was used to establish Km and Vmax. ANOVA with Dunnet's multiple range test was used to compare catalytic efficiencies between the FMO1 wild-type and H97Q, I303V, I303T, and R502X; p < 0.05 was the accepted level of significance.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of FMO Isoforms
The FMO1 and FMO3 variants were expressed in the baculovirus system (Figs. 2A and 2B). Expression levels of FMO1 and FMO3 variants were 0.26–0.66 nmol/mg microsomal protein. FMO1 levels also were measured in the supernatant and pellet at various stages of microsome preparation. Neither of the variants was detected in any other fraction but the microsomal fraction (immunoblot detection level: 20–30 pmol).



View larger version (92K):
[in this window]
[in a new window]
 
FIG. 2. Expression of human FMO1 variants. (A) SDS-PAGE. Microsomal proteins (20 µl) were separated on a 16% SDS-polyacrylamide gel and stained with coomassie blue. Concentration of microsomal protein is indicated in parenthesis. Lane 1, empty bacmid (2.01 mg/ml); Lane 2, FMO1 H97Q (3.51 mg/ml); Lane 3, FMO1 I303V (3.22 mg/ml); Lane 4, FMO1 I303T (2.47 mg/ml); Lane 5, FMO1 R502X (3.87 mg/ml); Lane 6, FMO1 wild-type (3.10 mg/ml). (B) Western blot of baculovirus expressed FMO1. SDS-PAGE immunoblot was carried out as described in Materials and Methods. Samples were normalized with regards to flavin content. Lane 1, empty bacmid; Lane 2, FMO1 H97Q; Lane 3, FMO1 I303V; Lane 4, FMO1 I303T; Lane 5, FMO1 R502X; Lane 6, FMO1 wild-type.

 
Catalytic Activity of FMO1 Variants
Kinetic parameters were calculated for the FMO1 variants using methimazole, imipramine, and methyl p-tolyl sulfide as substrates (Tables 3, 4 and 5). Additionally, thiourea oxidation was determined for wild type and R502X. NADPH oxidation in microsomes prepared from insect cells transfected with empty bacmid or mock transfected was low and set as baseline. In the methimazole NADPH oxidation assay, both the H97Q and I303T variants demonstrated higher Vmax and Km than the wild-type FMO1. Catalytic efficiency for the two variants was lowered (H97Q: 81%; I303T: 78%), but the difference was not statistically significant, due to higher Km values. A statistically significant difference between the I303V variant and the wild type also was not observed in this study (p > 0.05). R502X did not show catalytic activity when methimazole was used as a substrate but did demonstrate activity for thiourea, although with significantly reduced Vmax and Km (31 nmol/min/nmol flavin and 5 µM, p < 0.05). Vmax and Km values obtained for thiourea oxidation by wild-type FMO1 were 81.2 nmol/min/nmol flavin and 15 µM, respectively. N-oxygenation of imipramine by FMO1 corresponded relatively well to what has previously been observed (Stevens et al., 2003Go). Only minor changes in catalytic activity were observed for the FMO1 variants.


View this table:
[in this window]
[in a new window]
 
TABLE 3 S-Oxygenation of Methimazole by Baculovirus-Expressed FMO1 Variants

 

View this table:
[in this window]
[in a new window]
 
TABLE 4 N-Oxidation of Imipramine

 

View this table:
[in this window]
[in a new window]
 
TABLE 5 Kinetic Parameters and Stereochemistry of Methyl p-Tolyl Sulfide Sulfoxidation for Expressed FMO1 Variants

 
Stereochemistry of methyl p-tolyl sulfide sulfoxidation by FMO1 was similar to results determined previously (Sadeque et al., 1992Go). The only enantiomer observed was the (R)-sulfoxide. A peak marking the (S)-sulfoxide was not detected in any of the FMO1 assays. Product formation was linear for the first 30 min for all variants. Based on standard curves with the (R)- and (S)-sulfoxides, at least 95% of the sulfoxide formed was the (R)-enantiomer. Heat treatment in the absence of NADPH reduced FMO1 activity to less than 15% (data not shown). No difference was observed in heat lability of any of the variants examined. Human liver microsomes, obtained from Gentest, catalyzed the formation of a racemic 50% R/S mixture of the enantiomers (data not shown). The Vmax values determined for the variants followed the same trend as for the methimazole assay, with H97Q and I303T being elevated, and the I303V equal to the wild-type FMO1. Vmax/Km ratios for H97Q and I303T were found to be greater (p < 0.05) than the rest of the variants. The R502X variant was catalytically active, but with a lowered Vmax and Km relative to the other variants.

The FMO1 variants formed fenthion sulfoxide with a high level of stereoselectivity and with high catalytic efficiencies (Table 6). All variants formed the peak 1 (22 min retention time) enantiomer. Peak 1 was identified as the (+)-fenthion sulfoxide using circular dichroism (data not shown). FMO3 demonstrated much lower catalytic efficiency and stereoselectivity for fenthion than FMO1 (Table 7). Catalytic activity of the FMO3 D132H variant was not statistically different from the wild type.


View this table:
[in this window]
[in a new window]
 
TABLE 6 Kinetic Parameters and Stereochemistry of Fenthion Sulfoxidation for Expressed FMO1 Variants

 

View this table:
[in this window]
[in a new window]
 
TABLE 7 Kinetic Parameters and Stereochemistry of Fenthion Sulfoxidation for Expressed FMO3 Variants

 
Inhibition of Acetylcholinesterase by Fenthion and Fenthion Sulfoxide
Acetylcholinesterase activity was determined by the colorimetric method of Ellman et al. (1961)Go. Km for acetylthiocholine was determined to be 150 µM (Fig. 3). IC50 for fenthion and racemic fenthion sulfoxide at an acetylthiocholine concentration equal to Km was 0.015 and 0.26 mM, respectively. The (+)- and (–)-sulfoxides were equally strong inhibitors of acetylcholinesterase with IC50 similar to racemic fenthion sulfoxide.



View larger version (11K):
[in this window]
[in a new window]
 
FIG. 3. Inhibition of acetylcholinesterase by fenthion and fenthion sulfoxide. Purified recombinant human acetylcholinesterase was incubated with either fenthion (25 µM) or racemic fenthion sulfoxide (500 µM) before adding acetylthiocholine. IC50 values for fenthion and fenthion sulfoxide were 0.015 mM and 0.26 mM, respectively. No difference was observed between the fenthion sulfoxide stereoisomers.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The FMOs generally convert lipophilic heteroatom-containing xenobiotics to polar compounds that possess decreased toxic potential. An individual with impaired FMO activity may show exaggerated toxicity following exposure to a compound detoxified by FMO. It may also have clinical consequences, with potentially longer serum half-lives of FMO pharmaceutical substrates. In order to investigate the individual contribution of each amino acid to catalytic activity we chose to express these novel FMO1 variants using a baculovirus system and measure the oxidation of five substrates, fenthion, methimazole, methyl p-tolyl sulfide, imipramine, and thiourea. In addition to establishing the kinetic parameters, we wanted to identify possible FMO1 variants with altered stereoselectivity. This is especially important considering FMO1's previously reported high level of stereoselectivity (Sadeque et al., 1992Go). Studying the stereochemistry of sulfoxidation by FMO1 variants allows for the detection of subtle changes in the substrate binding site of the enzyme.

All genes encoding the variant proteins analyzed in this study were previously identified in African-Americans (Furnes et al., 2003Go). Lack of SNP frequencies from other ethnicities precludes any assumption of whether these variants are ethnic specific, although the I303V and I303T variants were recently observed in African-Americans but not in Caucasians (Hines et al., 2003Go). Neither the H97Q nor R502X variants were observed in that study, suggesting that they may be found with even less frequency in the African-American population than the I303V and I303T. One study, although with a very limited sample size, observed higher levels of kidney FMO1 in African-Americans than Caucasians (Krause et al., 2003Go). This may be due to a high frequency SNP (FMO1*6) found in the YY1 region of Hispanic-American samples (30%), but at a lower frequency in African-Americans (13%) (Hines et al., 2003Go). This SNP prevents binding of the YY1 transcription factor and is associated with a 2- to 3-fold loss of FMO1 promoter activity. Differences in FMO1 protein levels between individuals could cancel out or compound the differences observed in the catalytic efficiency of FMO1 variants. A few relevant high affinity substrates have been identified for FMO1, although none of them have yet been used as indicators of in vivo FMO1 catalytic activity. The cyclooxygenase inhibitor sulindac is converted to its sulfoxide (Hamman et al., 2000Go), and a metabolite of the alcohol deterrent disulfiram is oxidized to a sulfine (Pike et al., 2001Go) by FMO1 with high turnover numbers. N-oxygenation of the tricyclic antidepressant imipramine also has been associated with human FMO1 metabolism (Stevens et al., 2003Go).

The unique role of FMO1 as a major fetal liver-, adult kidney-, and intestinal-FMO isoform makes it important to predict any potentially adverse effects of having altered FMO1 activity. This also is compounded by the fact that the role of FMO1 in human drug metabolism, and fetal drug clearance in general, is not well elucidated. Several isoforms of P450 have been identified in the human kidney, but the overall contribution of the P450 system to drug metabolism in the kidney is thought to be quantitatively less compared to liver metabolism. Adverse effects due to altered FMO1 metabolism may be manifested differently in the fetus and adult due to the expression pattern.

Of the four FMO1 variants that were characterized, the wild-type histidine in the H97Q variant was the least conserved. A glutamine residue is found in both mouse and rat FMO1, and catalytic studies of the respective proteins (Itoh et al., 1993Go, 1997Go) indicate that they are catalytically active. The presence of a valine or isoleucine in all of the mammalian FMOs in the 303 position possibly suggests the requirement of a hydrophobic aliphatic amino acid in that position. A threonine in the 303 position introduces a polar hydroxyl group, possibly leading to changes in hydrogen bonding and/or tertiary structure. The R502 residue marks the last completely conserved amino acid in vertebrate FMOs and precedes a row of 20–30 hydrophobic amino acids. The truncated FMO1 protein was catalytically active and associated with the microsomal fraction, suggesting that the C-terminal hydrophobic section in FMO1 is not critical for catalytic activity nor determines membrane localization. The predominant FMO2 allele in Asians and Caucasians encodes a truncated protein lacking the last 64 amino acids rendering the enzyme inactive (Whetstine et al., 2000Go). Deletion analysis of FMO3 has revealed a significant reduction in catalytic efficiency for a 510X variant (Cashman et al., 2000Go). In contrast, Lawton and Philpot (1993)Go demonstrated that the 26 C-terminal amino acids of rabbit lung FMO2 could be deleted without affecting catalytic activity or subcellular location. The fact that the R502X, as well as other variants, retains its stereoselectivity with regards to methyl p-tolyl sulfide oxidation implies that the conformation of the substrate-binding pocket is still intact.

Flavin-containing monooxygenases have previously been shown to metabolize the organophosphate insecticide fenthion to its sulfoxide (Venkatesh et al., 1991Go). In rats, the racemic sulfoxide is only slightly more toxic than the parent compound (LD50250 vs. 325 mg/kg, single dose), and an almost equal prolonged inhibitory effect on cholinesterase is seen (Dubois and Kinoshita, 1964Go). The recombinant FMO1 variants stereoselectively formed the fenthion (+)-sulfoxide with surprisingly high Vmax values, while the FMO3 isoform formed only the sulfoxide with moderate turnover numbers and stereospecificity. The newly identified FMO3 D132H variant is so far only observed in African-Americans (Lattard et al, 2003Go) and was not associated with a statistically significant reduction of catalytic efficiency in this study. Although the sulfoxide is a weaker inhibitor of human acetylcholinesterase than fenthion, the complexity of metabolism and toxic effects precludes any conclusion that this is a detoxification pathway in humans. It is unclear whether sulfoxidation precedes oxon formation and whether the P450 responsible is stereoselective. The significance of producing only the (+) sulfoxide also remains to be seen.

In summary, this study is the first published report on the expression and catalytic activity of novel FMO1 variants. The overall conservation of FMO1 sequence and catalytic activity suggests that this is an important enzyme in humans. The current data available on FMO1 variants and catalytic activity indicate that FMO1 is active in all individuals. More studies on FMO1 SNPs could identify variants that cause interindividual differences in response to drugs and toxicants.


    ACKNOWLEDGMENTS
 
Bjarte Furnes was partially funded by a UC Toxic Substances Research and Teaching Program (UCTSR&TP) fellowship.


    NOTES
 

1 To whom correspondence should be addressed at 2217 Geology Bldg., University of California, Riverside, Riverside, CA 92521. Fax: (909) 787-3993. E-mail: bfurnes{at}citrus.ucr.edu. Reprint requests should be directed to Dr. Daniel Schlenk, 2217 Geology Bldg., University of California, Riverside, Riverside, CA 92521


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.[CrossRef][ISI][Medline]

Cashman, J. R. (1995). Structural and catalytic properties of the mammalian flavin-containing monooxygenase. Chem. Res. Toxicol. 8, 165–181.[ISI]

Cashman, J. R., Akerman, B. R., Forrest, S. M., and Treacy, E. P. (2000). Population-specific polymorphisms of the human FMO3 gene: Significance for detoxication. Drug. Metab. Dispos. 28, 169–173.[Abstract/Free Full Text]

Cashman, J. R., and Zhang, J. (2002). Interindividual differences of human flavin-containing monooxygenase 3: Genetic polymorphisms and functional variation. Drug. Metab. Dispos. 30, 1043–1052.[Abstract/Free Full Text]

Dubois, K. P., and Kinoshita, F. (1964). Acute toxicity and anticholinesterase action of O,O-dimethyl O-[4-(methylthio)-m-tolyl] phosphorothioate (DMTP; Baytex) and related compounds. Toxicol. Appl. Pharmacol. 6, 86–95.[ISI]

Ellman, G. L., Courtney, D. K., Andres, V., Jr., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–90.[CrossRef][ISI][Medline]

Francis, J. I., and Barnes, J. M. (1963). Studies on the mammalian toxicity of Fenthion. Bull. World Health Organ. 29, 205–212.[ISI][Medline]

Furnes, B., Feng, J., Sommer, S. S., and Schlenk, D. (2003). Identification of novel variants of the flavin-containing monooxygenase gene family in African Americans. Drug. Metab. Dispos. 31, 187–193.[Abstract/Free Full Text]

Hajjar, N. P., and Hodgson, E., (1980). Flavin adenine dinucleotide–dependent monooxygenase: Its role in the sulfoxidation of pesticides in mammals. Science 209, 1134–1136.[ISI][Medline]

Hamman, M. A., Haehner-Daniels, B. D., Wrighton, S. A., Rettie, A. E., and Hall, S. D. (2000). Stereoselective sulfoxidation of sulindac sulfide by flavin-containing monooxygenases. Comparison of human liver and kidney microsomes and mammalian enzymes. Biochem. Pharmacol. 60, 7–17.[CrossRef][ISI][Medline]

Hines, R. N., Luo, Z., Hopp, K. A., Cabacungan, E. T., Koukouritaki, S. B., and McCarver, G. (2003). Genetic variability at the human FMO1 locus: Significance of a basal promoter Yin Yang 1 element polymorphism (FMO1*6). J. Pharmacol. Exp. Ther. 306, 1210–1218.[Abstract/Free Full Text]

Itoh, K., Kimura, T., Yokoi, T., Itoh, S., and Kamataki, T. (1993). Rat liver flavin-containing monooxygenase (FMO): cDNA cloning and expression in yeast. Biochim. Biophys. Acta. 1173, 165–171.[ISI][Medline]

Itoh, K., Nakamura, K., Kimura, T., Itoh, S., and Kamataki, T. (1997). Molecular cloning of mouse liver flavin containing monooxygenase (FMO1) cDNA and characterization of the expression product: Metabolism of the neurotoxin, 1,2,3,4-tetrahydroisoquinoline (TIQ). J. Toxicol. Sci. 22, 45–56.[Medline]

Koukouritaki, S. B., Simpson, P., Yeung, C. K., Rettie, A. E., and Hines, R. N. (2002). Human hepatic flavin-containing monooxygenases 1 (FMO1) and 3 (FMO3) developmental expression. Pediatr. Res. 51, 236–243.[Abstract/Free Full Text]

Krause, R. J., Lash, L. H., and Elfarra, A. A. (2003). Human kidney flavin-containing monooxygenases and their potential roles in cysteine S-conjugate metabolism and nephrotoxicity. J. Pharmacol. Exp. Ther. 304, 185–191.[Abstract/Free Full Text]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[ISI][Medline]

Lattard, V., Zhang, J., Tran, Q., Furnes, B., Schlenk, D., Cashman, J. R. (2003). Two new polymorphisms of the FMO3 gene in Caucasian and African-American populations: Comparative genetic and functional studies. Drug. Metab. Dispos. 31, 854–860.[Abstract/Free Full Text]

Lawton, M. P., and Philpot, R. M. (1993). Functional characterization of flavin-containing monooxygenase 1B1 expressed in Saccharomyces cerevisiae and Escherichia coli and analysis of proposed FAD- and membrane-binding domains. J. Biol. Chem. 268, 5728–5734.[Abstract/Free Full Text]

Luo, Z., and Hines, R. N. (2001). Regulation of flavin-containing monooxygenase 1 expression by ying yang 1 and hepatic nuclear factors 1 and 4. Mol. Pharmacol. 60, 1421–1430.[Abstract/Free Full Text]

Miller, M. M., James, R. A., Richer, J. K., Gordon, D. F., Wood, W. M., and Horwitz, K. B. (1997). Progesterone regulated expression of flavin-containing monooxygenase 5 by the B-isoform of progesterone receptors: Implications for tamoxifen carcinogenicity. J. Clin. Endocrinol. Metab. 82, 2956–2961.[Abstract/Free Full Text]

Pike, M. G., Mays, D. C., Macomber, D. W., and Lipsky, J. J. (2001). Metabolism of a disulfiram metabolite, S-methyl N,N-diethyldithiocarbamate, by flavin monooxygenase in human renal microsomes. Drug. Metab. Dispos. 29, 127–132.[Abstract/Free Full Text]

Sadeque, A. J., Eddy, A. C., Meier, G. P., and Rettie, A. E. (1992). Stereoselective sulfoxidation by human flavin-containing monooxygenase. Evidence for catalytic diversity between hepatic, renal, and fetal forms. Drug. Metab. Dispos. 20, 832–839.[Abstract]

Schlenk, D., Cashman, J. R., Yeung, C., Zhang, X., and Rettie, A. E. (2002). Role of human flavin-containing monooxygenases in the sulfoxidation of [14C]aldicarb. Pestic. Biochem. Phys. 73, 67–73.[CrossRef][ISI]

Stevens, J. C., Melton, R. J., Zaya, M. J., and Engel, L. C. (2003). Expression and characterization of functional dog flavin-containing monooxygenase 1. Mol. Pharmacol. 63, 271–275.[Abstract/Free Full Text]

Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350–4354.[Abstract]

Treacy, E. P., Akerman, B. R., Chow, L. M., Youil, R., Bibeau, C., Lin, J., Bruce, A. G., Knight, M., Danks, D. M., Cashman, J. R., et al. (1998). Mutations of the flavin-containing monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication. Hum. Mol. Genet. 7, 839–845.[Abstract/Free Full Text]

Venkatesh, K., Levi, P. E., and Hodgson, E. (1991). The flavin-containing monooxygenase of mouse kidney. A comparison with the liver enzyme. Biochem. Pharmacol. 42, 1411–1420.[ISI][Medline]

Whetstine, J. R., Yueh, M. F., McCarver, D. G., Williams, D. E., Park, C. S., Kang, J. H., Cha, Y. N., Dolphin, C. T., Shephard, E. A., Phillips, I. R., and Hines, R. N. (2000). Ethnic differences in human flavin-containing monooxygenase 2 (FMO2) polymorphisms: Detection of expressed protein in African-Americans. Toxicol. Appl. Pharmacol. 168, 216–224.[CrossRef][ISI][Medline]

Wyatt, M. K., Overby, L. H., Lawton, M. P., and Philpot, R. M. (1998). Identification of amino acid residues associated with modulation of flavin-containing monooxygenase (FMO) activity by imipramine: Structure/function studies with FMO1 from pig and rabbit. Biochemistry 37, 5930–5938.[CrossRef][ISI][Medline]

Yeung, C. K., Lang, D. H., Thummel, K. E., and Rettie, A. E. (2000). Immunoquantitation of FMO1 in human liver, kidney, and intestine. Drug. Metab. Dispos. 28, 1107–1111.[Abstract/Free Full Text]

Ziegler, D. M. (1988). Flavin-containing monooxygenases: Catalytic mechanism and substrate specificities. Drug. Metab. Rev. 19, 1–32.[ISI][Medline]