Environmental Toxicology Program, University of California, Riverside, California 92521
Received November 25, 2003; accepted January 20, 2004
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ABSTRACT |
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Key Words: flavin-containing monooxygenase; fenthion; thiourea; methimazole; African-American; polymorphisms.
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INTRODUCTION |
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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., 2000). Lower levels of FMO1 also have been detected in the intestines. Considerable interindividual differences also are observed (Koukouritaki et al., 2002
). Hepatic FMO expression undergoes significant changes during development: FMO1 expression is highest in the embryo at 815 weeks gestation, and suppression occurs within 3 days after birth (Koukouritaki et al., 2002
). Onset of FMO3 expression is highly variable but is detectable in most individuals at the age of 12 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, and HNF4
have been identified in the major FMO1 human and rabbit promoter (Hines et al., 2003
; Luo and Hines, 2001
). 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., 1997
). 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, 2002). Functional characterization of these variants has been helpful in linking deficiency in FMO3 catalytic activity to the inborn disease trimethylaminuria (Treacy et al., 1998
). 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., 2003
). 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.
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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.
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MATERIALS AND METHODS |
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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.
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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 (1.01.3 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 89 followed by vigorous shaking for 7075 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 24 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, 1970; Towbin et al., 1979
). 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, 1976
). 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., 1998). The reaction mixture contained 75100 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 2.5250 µM were used in a total reaction volume of 1 ml. All incubations were carried out in triplicate. An NADPH extinction coefficient of 6220 M1 cm1 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 75100 µg microsomal protein, 1.3 mM NADPH, 3.3 mM MgCl2, and 11000 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) (07 min) that was slowly increased to 100% (720 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.
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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.
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RESULTS |
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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.
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DISCUSSION |
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All genes encoding the variant proteins analyzed in this study were previously identified in African-Americans (Furnes et al., 2003). 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., 2003
). 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., 2003
). 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., 2003
). 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., 2000
), and a metabolite of the alcohol deterrent disulfiram is oxidized to a sulfine (Pike et al., 2001
) by FMO1 with high turnover numbers. N-oxygenation of the tricyclic antidepressant imipramine also has been associated with human FMO1 metabolism (Stevens et al., 2003
).
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., 1993, 1997
) 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 2030 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., 2000
). Deletion analysis of FMO3 has revealed a significant reduction in catalytic efficiency for a 510X variant (Cashman et al., 2000
). In contrast, Lawton and Philpot (1993)
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., 1991). 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, 1964
). 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, 2003
) 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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
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