Flavin-containing monooxygenase activity in human liver microsomes

Human liver microsomal flavin-containing monooxygenase activity has been studied using dimethylaniline N-oxidation and thiobenzamide S-oxidation. Except for one subject, the capacity of human liver microsomes to mediate these reactions were markedly increased at pH 8.4 compared to pH 7.4. The mean dimethylaniline N-oxidase activities at pH 7.4 and 8.4 in the four subjects tested were 2.49 +/- 1.13 and 6.59 +/- 4.04 nmol mg-1 min-1, respectively (mean +/- SD, N = 4). The mean thiobenzamide S-oxidase activities at pH 7.4 and 8.4 were 1.39 +/- 0.51 and 2.74 +/- 1.28 nmol mg-1 min-1, respectively. At pH 7.4, an antibody to the human liver NADPH-cytochrome P-450 reductase inhibited dimethylaniline N-oxidation between 4 and 38%. The same antibody had no effect on this reaction at pH 8.4. Except for one subject, a battery of cytochrome P-450 inhibitors also had little effect on this reaction. Further, preincubating human microsomes at 45 degrees C in the absence of NADPH for 4 min destroyed approximately 90% of the dimethylaniline N-oxidase activity. These data collectively suggested that the flavin-containing mono-oxygenase is the major enzyme mediating this reaction in human liver microsomes. In contrast to dimethylaniline N-oxidation, thiobenzamide S-oxidation was significantly inhibited by the anti-reductase at both pH 7.4 and 8.4, respectively. These data indicate that cytochromes P-450 contribute significantly to this reaction in human liver microsomes.     

Oxidation of thiobenzamide by the FAD-containing and cytochrome P-450-dependent monooxygenases of liver and lung microsomes

Two distinct microsomal pathways involved in the metabolism of thiobenzamide to thiobenzamide S-oxide have been identified and quantitated in the liver and lungs of mice and rats, using a highly inhibitory antibody against NADPH-cytochrome P-450 reductase. Approximately 50 and 65% of the oxidation in mouse and rat liver microsomes, respectively, was due to the FAD-containing monooxygenase, the remainder being catalyzed by cytochrome P-450. In the mouse lung, S-oxidation was predominantly via the FAD-containing monooxygenase while that in the rat lung was about 60% via the FAD-containing enzyme and 40% via cytochrome P-450. Cytochrome P-450-dependent S-oxidation of thiobenzamide was induced in the liver by treatment of mice with phenobarbital and slightly increased by treatment with 3-methylcholanthrene, while in rat liver either of these treatments caused only a small increase in metabolism due to cytochrome P-450. Thermal inactivation of the FAD-containing monooxygenase left the cytochrome P-450 component essentially unchanged. Thermally treated microsomes had a pH activity profile characteristic of cytochrome P-450 and were less inhibited by methimazole and thiourea when compared to untreated microsomes. Female mouse liver microsomes had a much higher, and female rat liver microsomes a lower, ability to S-oxidize thiobenzamide when compared to the males.     

The role of flavin-containing monooxygenase in the N-oxidation of the pyrrolizidine alkaloid senecionine

The pyrrolizidine alkaloid, senecionine, is N-oxidized by purified pig liver flavin-containing monooxygenase but not by purified rabbit lung flavin-containing monooxygenase. The activity of the pig liver enzyme toward senecionine was linear with time and amount of enzyme. The oxygenation was not due to some indirect mechanism, such as O2- release from the enzyme, as scavengers of activated oxygen had no effect on product formation. The Km of purified pig liver flavin-containing monooxygenase for senecionine was 0.3 mM. Although senecionine is a substrate for the pig liver enzyme, studies performed with rat liver microsomes suggest that, in this species, cytochromes P-450 catalyze the majority of senecionine-N-oxidation. These experiments included inhibition by chemical inhibitors of P-450, treatment of the microsomes with elevated temperatures, inhibition by anti-NADPH-cytochrome P-450 reductase antibody, the effect of dexamethasone on N-oxidation, and relative amounts of flavin-containing monooxygenase determined by immunoquantitation. These results demonstrate that flavin-containing monooxygenase can be involved in the detoxication of pyrrolizidine alkaloids via N-oxidation, but the relative contribution of flavin-containing monooxygenase and cytochromes P-450 may be species and tissue dependent.     

Suppression of flavin-containing monooxygenase by overproduced nitric oxide in rat liver.

Effects of excessive nitric oxide (NO) produced in vivo by an i.p. injection of bacterial lipopolysaccharide (LPS) on hepatic microsomal drug oxidation catalyzed by flavin-containing monooxygenase (FMO) were determined. At 6 and 24 h after the LPS injection, liver microsomes were isolated and FMO activities were determined by using FMO substrates like thiobenzamide, trimethylamine, N,N-dimethylaniline, and imipramine. Liver microsomal FMO activities of LPS-treated rats were decreased significantly for all these substrates. Microsomal content of FMO1 (the major form in rat liver) in LPS-treated rats as determined by immunoblotting, was severely decreased as well. In support of this, hepatic content of FMO1 mRNA was decreased by 43.6 to 67.3%. However, the hepatic content of inducible NO synthase (iNOS) mRNA was increased by 2.6- to 5.4-fold and the plasma nitrite/nitrate concentration was increased by about 30-fold in the LPS-treated rats. When this overproduction of NO in the LPS-treated rats was inhibited in vivo by a single or repeat doses of either a general NOS inhibitor N(G)-nitro-L-arginine or a specific iNOS inhibitor aminoguanidine, the FMO1 mRNA levels were not severely depressed (70-85% of the control level). Attendant with the reduction of plasma nitrite/nitrate concentration by single and repeated doses of NOS inhibitors, activity and content of FMO1 in liver microsomes isolated from these NOS inhibitor cotreated rats were restored partially (in single-dose inhibitors) or completely (in repeat doses). In contrast to these NO-mediated in vivo suppressive effects on the mRNA and enzyme contents of FMO1 as well as the FMO activity, the NO generated in vitro from sodium nitroprusside did not inhibit the FMO activities present in microsomes of rat and rabbit liver as well as those present in rabbit kidney and lung. Combined, the excessive NO produced in vivo (caused by the LPS-dependent induction of iNOS) suppresses the FMO1 mRNA and enzyme contents as well as the FMO activities without any direct in vitro effect on the activities of premade FMO enzyme. These findings suggest that NO is an important mediator involved in the suppression of FMO1 activity in vivo. Thus, together with the previously reported suppression on the cytochrome P-450 activities, the overproduced NO in the liver caused by induction of iNOS under conditions of endotoxemia or sepsis suppresses FMO and appears to be responsible for the decreased drug oxidation function observed generally under conditions of systemic bacterial or viral infections.     

Tissue- and species-dependent expression of multiple forms of mammalian microsomal flavin-containing monooxygenase

The immunochemical relatedness, multiplicity, and expression of microsomal flavin-containing monooxygenases were assessed in hepatic, pulmonary, and renal microsomal preparations from adult, male rabbits, mice, rats, guinea pigs, and hamsters. Preparations from adult female (pregnant and nonpregnant) and immature male and female rabbits were also examined. Microsomes were analyzed by immunoblotting with polyclonal antibodies to flavin-containing monooxygenases purified from pig, mouse, and rabbit liver, and rabbit lung. Pulmonary flavin-containing monooxygenases, which differ from the major forms of the enzyme present in liver, were detected in pulmonary samples from all species. The major hepatic enzymes, or very closely related proteins, were also detected in pulmonary samples from every species except the rabbit. Three forms of pulmonary flavin-containing monooxygenase are found in rabbit and guinea pig. Differences in the expression of these three forms were observed with rabbits; all three were detected with some individuals and only two with others. The hepatic form of the flavin-containing monooxygenase is present in kidney of all species examined, and the pulmonary form is detected in kidney of rabbits, mice, and hamsters, but not rats or guinea pigs. Kidneys and lungs of individual rabbits were of the same phenotype with respect to the expression of the pulmonary forms of the enzyme. The findings show that the expression of flavin monooxygenase isozymes is tissue and species dependent.     

Activities of cytosolic and microsomal drug oxidases of rat hepatocytes in primary culture

Sensitive and specific chromatographic assays for the measurement of flavin-containing monooxygenase (N-oxygenase and S-oxygenase activities) and aldehyde oxidase activities in rat hepatocyte primary cultures were developed. Conditions for the measurement of enzymatic activities in rat liver cell cultures were first optimized using freshly isolated cell suspensions. Activities of the cytochrome P-450/P-448 isozymes in rat hepatocytes maintained in primary culture [assessed by the O-deethylation of 7-ethoxycoumarin (P-450/P-448) and the N-demethylation of N,N-dimethylaniline (P-450)] rapidly declined to 25% of the initial levels by 48 hr in culture. The flavin-containing monooxygenase system was considerably more stable in cell culture. Flavin N-oxygenase activity (assessed by the N-oxidation of N,N-dimethylaniline) declined slightly (10-15%) and remained almost constant over the 48-hr culture period, whereas S-oxygenase activity (assessed by the S-oxygenation of tetrahydrothiophen) gradually declined and stabilized at approximately 65% of its initial activity at 48 hr in culture. Aldehyde oxidase activity (assessed by the 1-hydroxylation of phthalazine) declined to approximately 20% of the initial value by 48 hr in culture. The differential stability of the microsomal and cytosolic drug oxidases in rat hepatocytes in primary culture demonstrates some of the limitations of this model for metabolic studies.     

Rabbit lung flavin-containing monooxygenase. Purification, characterization, and induction during pregnancy

A flavin-containing monooxygenase has been purified to apparent homogeneity from lung microsomes of pregnant rabbits and characterized with respect to a number of physical and catalytic parameters. The apparent molecular weight, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 59,000, and the lung microsomal flavoprotein was shown to contain 14 nmol of FAD/mg of protein. Addition of NADP+ to the oxidized flavoprotein produced a shift in the spectrum characteristic of the flavin-containing monooxygenase from porcine liver, and addition of small amounts of NADPH to the oxidized rabbit lung enzyme produced a stable spectral intermediate consistent with that of a 4a-peroxyflavin. Rabbit lung flavin-containing monooxygenase differed markedly from the porcine liver enzyme in exhibiting a broader pH optimum from 8.5-10.5, by not being inhibited by concentrations of sodium cholate as high as 1% and by withstanding, in the absence of NADPH, incubation at 45 degrees for at least 10 min with no significant loss of activity. Unlike the pig liver enzyme, purified rabbit lung enzyme was not activated by n-octylamine and, in fact, n-octylamine stimulated NADPH oxidation. A number of compounds known to be substrates of the pig liver enzyme, including benzphetamine, chlorpromazine, and imipramine, are not substrates for the rabbit lung enzyme, whereas prochlorperazine and trifluoperazine are excellent substrates. Antibodies to rabbit lung flavin-containing monooxygenase were raised in guinea pig and utilized for the immunoquantitation of this enzyme throughout gestation. The activity (as determined by N,N-dimethylaniline-N-oxidation) and amount of rabbit lung flavin-containing monooxygenase were maximally induced (5-fold) on the 28th day of gestation. Liver microsomes from rabbit did not contain any of the lung form of flavin-containing monooxygenase at any time during gestation, as evidenced by results from Western blotting. These results demonstrate that, at least in rabbit, flavin-containing monooxygenase can exist as more than a single form. The physiological significance of the induction of this enzyme during pregnancy is not known.     

Enantioselective S-oxygenation of para-methoxyphenyl-1,3-dithiolane by various tissue preparations: effect of estradiol

Liver, kidney, and lung microsomes prepared from nonpretreated female Sprague-Dawley rats catalyze the NADPH- and oxygen-dependent S-oxygenation of para-methoxyphenyl-1,3-dithiolane. Studies on the biochemical mechanism of dithiolane S-oxygenation in liver, kidney, and lung microsomes suggest that this reaction is catalyzed in a diastereoselective and enantioselective fashion by the flavin-containing monooxygenase and, to a lesser extent, the cytochromes P-450. This conclusion is based on results examining the effects of selective cytochrome P-450 inhibitors and positive effectors, microsome heat-inactivation treatment, and alternate substrates for the flavin-containing monooxygenase. Liver and kidney microsomes prepared from ovarectomized female rats tended to have decreased S-oxygenase activity, compared with nonpretreated female rats, whereas ovarectomized rats pretreated with estradiol had markedly lower S-oxygenase activity. In contrast, lung microsomal S-oxygenase activity, which is low in pulmonary microsomes from nonpretreated female rats, increases 2-4-fold after ovariectomization and estradiol pretreatment. In female Sprague-Dawley rats, estradiol pretreatment is mainly responsible for the large decrease (or increase) in S-oxygenase activity observed in the tissues examined, although it is unlikely that estradiol alone controls flavin-containing monooxygenase S-oxygenase activity.     

Role of flavin-containing monooxygenase in the in vitro biotransformation of aldicarb in rainbow trout (Oncorhynchus mykiss).

1. The in vitro biotransformation of 14C-aldicarb was examined in liver, kidney, and gill microsomes from the rainbow trout (Oncorhynchus mykiss). 2. In all tissues the major metabolite was aldicarb sulphoxide. Addition of the cytochrome P-450 inhibitor, N-benzylimidazole, failed to alter significantly aldicarb sulphoxide levels, while co-incubation with the flavin-containing monooxygenase substrates, N,N-dimethylaniline or methimazole, caused significant decreases in sulphoxide formation in liver and gill microsomes. 3. Aldicarb sulphoxide formation was optimal at pH 8.0, and had Michaelis-Menten kinetics with an apparent Km of 46.7 microM and a Vmax of 0.216 nmol/min per mg. 4. Aldicarb sulphoxide formation was competitively inhibited by co-incubation with N,N-dimethylaniline in liver microsomes. These data indicate that flavin-containing monooxygenase plays an important role in the in vitro biotransformation of aldicarb in rainbow trout.     

Role of flavin-containing monooxygenase in the in vitro biotransformation of aldicarb in rainbow trout (Oncorhynchus mykiss)

1. The in vitro biotransformation of¹⁴C-aldicarb was examined in liver, kidney, and gill microsomes from the rainbow trout (Oncorhynchus mykiss).

2. In all tissues the major metabolite was aldicarb sulphoxide. Addition of the cytochrome P-450 inhibitor, N-benzylimidazole, failed to alter significantly aldicarb sulphoxide levels, while co-incubation with the flavin-containing monooxygenase substrates, N,N-dimethylaniline or methimazole, caused significant decreases in sulphoxide formation in liver and gill microsomes.

3. Aldicarb sulphoxide formation was optimal at pH8˙0, and had Michaelis-Menten kinetics with an apparent K_m of 46˙7 uM and a V_max of 0˙216nmol/min per mg.

4. Aldicarb sulphoxide formation was competitively inhibited by co-incubation with N,N-dimethylaniline in liver microsomes. These data indicate that flavin-containing monooxygenase plays an important role in the in vitro biotransformation of aldicarb in rainbow trout.     

Stereoselective S-oxygenation of 2-aryl-1,3-dithiolanes by the flavin-containing and cytochrome P-450 monooxygenases

The reaction of NalO4, highly purified flavin-containing monooxygenase (EC 1.14.13.8), and microsomes from hog liver with 2-aryl-1,3-dithiolanes and 2-aryl-1,3-dithiolane S-oxides was investigated. The initial rates determined for the microsome- and purified flavin-containing monooxygenase-catalyzed rate of S-oxidation of para-substituted 2-aryl-1,3-dithiolanes were similar, demonstrating that S-oxidation of these substrates occurred with similar velocities at saturating concentrations of substrate and, at least for the first S-oxidation, the reaction was insensitive to the nature of the para-substituent. The diastereoselectivity of S-oxygenation of 2-aryl-1,3-dithiolanes was determined and, in general, a marked preference for addition of oxygen to the sulfide sulfur atom was observed to occur trans to the aryl groups. In all cases examined, enantioselective enzymatic S-oxidation was observed. For S-oxide formation in microsomes, the data provided evidence for a minor role of cytochrome P-450 in S-oxide formation, but the flavin-containing monooxygenase was mainly responsible for production of S-oxide. In contrast to previous reports, the enantioselectivity of S-oxidation catalyzed by highly purified cytochrome P-450IIB-1 and cytochrome P-450IIB-10 was not always opposite to that catalyzed by hog liver flavin-containing monooxygenase activity. 2-Aryl-1,3-dithiolane S-oxides were also oxidized a second time by NalO4, microsomes, or highly purified flavin-containing monooxygenase from hog liver but not cytochrome P-450IIB-1 or P-450IIB-10. The rate of the second oxidation was 10-15-fold slower than the corresponding first S-oxidation and S,S'-dioxide formation was markedly dependent on the electronic nature of the para-substituent (Hammett correlation rho value of -1.3 and -1.1 for microsomes and highly purified flavin-containing monooxygenase from hog liver, respectively). The large dependence of the rate of S,S'-dioxide formation on the nature of the para-substituent demonstrates that velocity values at saturating concentrations of S-oxide were not the same for all 2-aryl-1,3-dithiolane S-oxides and suggests that the chemical nature of the 2-aryl-1,3-dithiolane S-oxide contributes to the rate-determining step of this enzymatic reaction.     

Physiological factors affecting the expression of FMO1 and FMO3 in the rat liver and kidney

FMO1 and FMO3, the main FMOs described in the rat, are highly expressed in the liver and the kidney. The age, from 3 to 11 weeks, and gender-dependent expression of FMO1 and FMO3 in the rat liver and kidney were investigated. Based on the enzyme activities, protein levels and mRNA levels, this study demonstrates an important increase in the expression of the FMO3 in the liver of male rats during a period that corresponds to the acquisition of the sexual maturity. Rat liver FMO1 remains unchanged during this period of observation. The evolutions of both isoforms in the kidney of the male rat are similar to those observed in the liver. On the contrary, the important decrease in the total flavin-containing monooxygenase (FMO) activity observed in the liver of female rat is linked to a considerable decrease in the FMO1-dependent activity, FMO1 protein and FMO1 mRNA levels as a function of age. The expression of the FMO3 in the liver does not seem to be affected by the age of the female rat. Inversely, the expression of FMO1 in the female rat kidneys does not seem to be modified as a function of age while the expression of FMO3 is strongly increased.     

Metabolism of antiparkinson agent dopazinol by rat liver microsomes

Metabolism of dopazinol (DZ) by liver microsomes from control and phenobarbital- and 3-methylcholanthrene-treated rats has been investigated. Liver microsomes from control and treated rats metabolized DZ to N-despropyl-DZ (39-53% of total metabolites); 8-hydroxy-DZ, a catechol metabolite (32-39%); and 5- or 6-hydroxy-DZ (12-20%). The last metabolite was identified as its dehydration product 5,6-dehydro-DZ. N-Dealkylation was favored only slightly over catechol formation (ratio = 1.2) by liver microsomes from control and phenobarbital-treated rats, whereas with liver microsomes from 3-methylcholanthrene-treated rats, N-dealkylation predominated (ratio = 1.7). Liver microsomes from control rats metabolized DZ at a rate of 0.86 nmol/nmol cytochrome P-450/min. Pretreatment of rats with phenobarbital or 3-methylcholanthrene stimulated rates of metabolism by 2.4- and 3-fold, respectively. Metabolism of DZ was inhibited by SKF 525-A, methimazole, and thiobenzamide. SKF 525-A completely inhibited metabolism of DZ, while methimazole and thiobenzamide, two alternate substrates of the microsomal flavin-containing monooxygenase (MFMO) inhibited N-dealkylation only. These results indicated that while the cytochrome P-450-dependent monooxygenase is the primary enzyme system in DZ oxidation, the MFMO also catalyzes the N-dealkylation reaction. The catechol metabolite was converted to isomeric O-methylated derivatives in approximately 1:1 ratio by purified catechol-O-methyl transferase or 105,000g liver cytosol. The late eluting isomer was 8-methoxy-DZ.     

Oxidation of N-hydroxynorzimeldine to a stable nitrone by hepatic monooxygenases

Liver microsomes from uninduced hogs catalyzed the NADPH-dependent N-oxidation of the hydroxylamine (Z)-3-(4-bromophenyl)-N-hydroxy-N-methyl-3-(3-pyridyl)allylamine. The conjugated nitrone N-[(2Z)-3-(4-bromophenyl)-3-(3-pyridyl)-2-propenylidene]methylamin e N-oxide was the principal product and accounted for 90-95% of the total hydroxylamine metabolized by microsomes. The nitrone had a relatively high chemical stability which was investigated at various conditions. Studies of the biochemical mechanisms for N-oxidation of N-hydroxy-norzimeldine showed that the reaction was catalyzed by the flavin-containing monooxygenase. This conclusion was based on studies with the purified hog liver flavin-containing monooxygenase and hog liver microsomes. Purified rat liver cytochrome P-450IIB-1 was ineffective at catalyzing N-oxidation of N-hydroxynorzimeldine.     

Effects of hypophysectomy on acetylcholinesterase and butyrylcholinesterase in the rat

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were examined in several tissues of normal and hypophysectomized male and female rats. Significant sex differences in the mean AChE activities of normal rats were observed in the superior cervical ganglion (three times more activity in males) and in serum (50% more activity in females). Sex differences in the BuChE activity of serum and liver were even larger (ten times more activity in females), but the activity of other tissues was similar in both sexes. Hypophysectomy had little effect on the mean activity of AChE but did alter BuChE activity in certain tissues. Most of the effects of hypophysectomy on mean BuChE activity were opposite in direction in the two sexes. For example, in males hypophysectomy caused increases in the BuChE activity of serum (300%) and liver (43%), while in females it caused decreases in both tissues (25 and 30% respectively). In rats of a given group, the AChE activity of each tissue appeared to be regulated independently of the activity in other tissues. By contrast, BuChE activity showed statistically significant correlations in more than half of the tissue-pairs examined in control rats of either sex. These correlations can be considered to reflect a tendency toward body-wide regulation. In female rats, the cross-tissue correlations were largely eliminated by hypophysectomy. This finding indicates that the regulation of BuChE mav be strongly affected by hormones under the control of the pituitary gland. However, in male rats, only the correlations involving atria were altered by hypophysectomy. Therefore, the effects of hormones on BuChE are probably both sex and tissue dependent.     

Differential hormonal regulation of carbonyl reductase activities in liver and kidney microsomes of rat

1. In the male rat, hepatic microsomal carbonyl reductase (CR) activity decreased by testectomy (Tx) was restored to the control level by the treatment with testosterone propionate (TP), even though the enzyme activity decreased by hypophysectomy (Hx) was not increased by the treatment with TP. On the other hand, renal microsomal CR activities decreased by Tx and Hx were markedly increased by the treatment with TP. 2. The treatment with TP had no effect on the CR activity in liver microsomes of the ovariectomized or hypophysectomized female rat. On the other hand, the CR activities in kidney microsomes of the ovariectomized and hypophysectomized female rat were significantly increased by the treatment with TP. 3. The results indicate that in rat programmed by neonatal androgens, the hepatic microsomal CR activity is regulated indirectly by androgens through the hypothalamus-pituitary system, whereas the hormonal regulation of the renal microsomal CR activity is not via the pituitary. We conclude that the regulatory mechanism of the CR activity in liver microsomes is distinguishable from that in kidney microsomes.     

Differential hormonal regulation of carbonyl reductase activities in liver and kidney microsomes of rat

1. In the male rat, hepatic microsomal carbonyl reductase (CR) activity decreased by testectomy (Tx) was restored to the control level by the treatment with testosterone propionate (TP), even though the enzyme activity decreased by hypophysectomy (Hx) was not increased by the treatment with TP. On the other hand, renal microsomal CR activities decreased by Tx and Hx were markedly increased by the treatment with TP. 2. The treatment with TP had no effect on the CR activity in liver microsomes of the ovariectomized or hypophysectomized female rat. On the other hand, the CR activities in kidney microsomes of the ovariectomized and hypophysectomized female rat were significantly increased by the treatment with TP. 3. The results indicate that in rat programmed by neonatal androgens, the hepatic microsomal CR activity is regulated indirectly by androgens through the hypothalamus-pituitary system, whereas the hormonal regulation of the renal microsomal CR activity is not via the pituitary. We conclude that the regulatory mechanism of the CR activity in liver microsomes is distinguishable from that in kidney microsomes.     

Compound heterozygosity for missense mutations in the flavin-containing monooxygenase 3 (FM03) gene in patients with fish-odour syndrome

Fish-odour syndrome is a highly unpleasant disorder of hepatic trimethylamine (TMA) metabolism characterized by a body odour reminiscent of rotting fish, due to excessive excretion of the malodorous free amine. Although fish-odour syndrome may exhibit as sequelae with other conditions (e.g. liver dysfunction), many patients exhibit an inherited, more persistent form of the disease. Ordinarily, dietary-derived TMA is oxidized to the nonodorous N-oxide by hepatic flavin-containing monooxygenase 3 (FMO3). Our previous demonstration that a mutation, P153L (C to T), in the FMO3 gene segregated with the disorder and inactivated the enzyme confirmed that defects in FMO3 underlie the inherited form of fish-odour syndrome. We have investigated the genetic basis of the disorder in two further affected pedigrees and report that the three propositi are all compound heterozygotes for causative mutations of FMO3. Two of these individuals possess the P153L (C to T) mutation and a novel mutation, N61S (A to G). The third is heterozygous for novel, M4341 (G to A), and previously reported, R492W (C to T), mutations. Functional characterization of the S61, 1434 and W492 variants, via baculovirus-mediated expression in insect cells, confirmed that all three mutations either abolished, or severely attenuated, the capacity of the enzyme to catalyse TMA N-oxidation. Although 1434 and W492 were also incapable of catalysing the S-oxidation of methimazole, S61 was fully active with this sulphur-containing substrate. Since an asparagine is conserved at the equivalent position to N61 of FMO3 in mammalian, yeast and Caenorhabditis elegans FMOs, the characterization of the naturally occurring N61S (A to G) mutation may have identified this asparagine as playing a critical role specifically in FMO-catalysed N-oxidation.     

Ascorbic acid deficiency and the flavin-containing monooxygenase

Activity of the flavin-containing monooxygenase (FMO) was reduced significantly in ascorbic acid deficient guinea pigs. Reduction in oxidation of dimethylaniline (DMA) and of thiobenzamide was associated with a decrease in the activity of the FMO. In both ascorbate supplemented and deficient guinea pig hepatic 12,000 g supernatant fractions, SKF-525A and n-octylamine did not inhibit DMA N-oxidation. Phenobarbital pretreatment did not increase the rate of N-oxidation of DMA. In addition, hepatic supernatant fractions thermally treated at 50 degree were unable to N-oxidize DMA, but 80% of the cytochrome P-450 activity was retained. Also, N-oxidation of DMA was reduced by 53% at pH 7.0, while oxidation of cytochrome P-450 specific substrates was inhibited by only 19%. Kinetic studies of DMA N-oxidation indicate no significant change in the apparent Km in ascorbate supplemented or deficient animals. The in vitro addition of ascorbic acid had no effect on the activity of the FMO. The toxicological implications of the reduction in FMO activity in ascorbic acid deficiency are discussed.     

Species, organ and cellular variation in the flavin-containing monooxygenase

The distribution of the flavin-containing monooxygenase (EC1.14.13.8) (FMO) between species, organs and cell types is summarized with particular reference to the organ specific forms present in mammalian lung and liver. The role of the FMO relative to cytochrome P-450 in the oxidation of the sulfur atoms of organosulfur compounds is considered with particular reference to the hepatatoxicant thiobenzamide, the insecticide phorate and the drug, thioridazine. Of special interest is the relative role of these enzymes in complex metabolic pathways of xenobiotics.