• Toxicological Research
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• 제17권
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• pp.127-133
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• 2001

Together with cytochrome P450 (CYP), flavin-containing monooxygenase (FMO) present in liver microsomes oxidizes various endogenous and exogenous chemicals. In an effort to determine the human FMO activity, we have developed two non-invasive urine analysis methods using caffeine (CA) and ranitidine (RA) as the probe compounds. As the production of theobromine (TB) and ranitidine N-oxide (RANO) from CA and RA is catalyzed primarily by the hepatic FMO, we have assigned the urinary molar ratios of TB/CA and RA/RANO as the in vivo FMO activity. In 200 age-matched Korean volunteers, the obtained TB/CA ratio ranged from 0.4 to 15.2 (38-fold difference) and the RA/RANO ratio from 5.7 to 27.2 (4.8-fold). The FMO activity of 20's, determined by caffeine metabolism, was the highest (2.5$\pm$l.9) and those of 30's, 40's, 50's, 60's and 70's were 40%, 50%, 24%, 39% and 36% of the 20's, respectively. Intake of grapefruit juice, known to contain flavonoids, inhibited the in vivo FMO (TB/CA) activity by 79%. Addition of the flavonoids like naringin, quercitrin and kaempferol, present in grapefruit juice, to the in vitro microso-mal FMO assay, thiobenzamide S-oxidation, produced 75%, 70% and 60% inhibition, respectively. Obtained Ki values of quercitrin, kaempferol and naringin on the in vitro FMO activity were 6.2, 12.0 and 13.9 $\mu\textrm{M}$, respectively. This suggested that the dose of drug should need to be adjusted to suit the individual FMO activities when the drugs metabolized by FMO are given to patients. As the intake of grapefruit juice has been identified to inhibit the FMO as well as CYP3A4 and lA2 activities, patients taking drugs metabolized by these enzymes should not drink grapefruit juice as the carrier.

• Molecular & Cellular Toxicology
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• 제4권1호
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• pp.5-10
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• 2008

The effect of obesity on the drug-metabolizing enzymes remains an important issue for clinician since obesity is a world wide epidemic problem. However, little is known about the effects of obesity on flavincontaining monooxygenase (FMO) production and activity. We show here for the first time that in vivo FMO activity determined by urinary ranitidine (RA) metabolites ratio in human, was higher in subjects with a high body mass index (BMI, kg/$m^2$, 21.97-30.32) than in those with an intermediate BMI (range 19.38-21.83). Moreover, there was a significant correlation between FMO activity and BMI in 209 subjects. In high fat diet-induced obese mice, we also observed that the hepatic expression of FMO (225% of lean mice) and the activity measured by the RA Noxidation rate ($513{\pm}58.1$ vs. $349{\pm}66.0$ pmol/hr per mg protein) were significantly higher than in lean mice fed a control diet. Unknown factors rather than leptin or insulin appeared to regulate the hepatic FMO production. Thus, FMO activity may be increased in obese or overweight individuals. Moreover, the regulation of FMO activity in subjects with morbid obesity, with or without complications and its clinical implications, should be investigated further.

• The Korean Journal of Physiology and Pharmacology
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• 제7권3호
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• pp.157-162
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• 2003

Our earlier studies found a significant correlation between the activities of ranitidine N-oxidation catalyzed by hepatic flavin-containing monooxygenase (FMO) and the presence of mutations in exon 4 (E158K) and exon 7 (E308G) of the FMO3 gene in Korean volunteers. However, caffeine N-1 demethylation (which is also partially catalyzed by FMO) was not significantly correlated with these FMO3 mutations. In this study, we examined another common mutation (V257M) in exon 6 of FMO3 gene. The V257M variant, which is caused by a point mutation (G769A), was commonly observed (13.21% allele frequency) in our subjects (n=159). This point mutation causes a substitution of $Val^{257}$ to $Met^{257}$, with transformation of the secondary structure. The presence of this mutant allele correlated significantly with a reduction in caffeine N-1-demethylating activity, but was not correlated with the activity of N-oxidation of ranitidine. In a family study, the low FMO activity observed in a person heterozygous for a nonsense mutation in exon 4 (G148X) and heterozygous for missense mutation in exon 6 (V257M) of FMO3 was attributed to the mutations. Our results suggest that various point mutations in the coding regions of FMO3 may influence FMO3 activity according to the probe substrates of varying chemical structure that correlate with each mutation on the FMO3 gene.

• The Korean Journal of Physiology and Pharmacology
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• 제1권5호
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• pp.591-595
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• 1997

An ex vivo assay determining the flavin-containing monooxygenase (FMO) activity in perfused rat liver has been developed by assessing the rate of thiobenzamide S-oxide (TBSO) formation from the infused thiobenzamide (TB). The hepatotoxicity by TB or TBSO was not a critical factor for maintaining the FMO activity for up to 50 min. The FMO activity expressed in nmoles TBSO produced/g liver/min was the same for the recycling and non-recycling perfusion. This implies that reduction of the oxidized TBSO back to the parent compound (TB) is negligible. Hydrolysis of the collected perfusates with either ${\beta}-glucuronidase$ or arylsulfatase did not increase the TBSO level and thus, TBSO does not appear to undergo conjugation either to glucuronide or sulfate esters. Thus, measuring the rate of TB S-oxidation in the isolated perfused liver with 1 mM TB for 50 min provides a useful tool for evaluation of the hepatic FMO activity in the absence of hepatic necrosis and without the interferences caused by further conjugation or back reduction of the TBSO to the parent TB.

• Environmental Analysis Health and Toxicology
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• 제15권4호
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• pp.139-145
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• 2000

FMO (Flavin-containing Monooxygenase, EC1.14.13.8)는 다양한 종류의 식품, 약물이나 기타 외래 유래물질(xenobiotics)을 산화시키는 NADPH와 $O_2$의존성 약물대사 효소이다 현재까지 5종의 subfamility가 존재하는 것으로 보고되어지고 있으며 그 중 가장 잘 알려진 FMO3는 대표적인 subfamility로서 주로 간에 존재한다. 사람 FMO에 관한 연구는 최근들어 활성화되기 시작했으며 질소, 황이나 인 등을 포함하는 친핵성 (nucleophilic)화합물이 대표적인 기질로 보고되어 있다. 본 연구에서는 항 산화작용이 있는 것으로 알려진 selenium을 포함하고 있는 화합물에 대한 사람의 FMO3의 기질특이성을 알아보고자 하였다. 사람 FMO3를 baculovirus system을 이용하여 발현시킨 후 그 microsomal FMO3을 이용하여 thiochline assay를 시행하였다. 그 결과 기질의 크기에 따라 활성의 차이가 있었으며 크기가 작은 selenium화합물은 기존의 질소나 인 등을 포함하는 기질보다 더 낮은 $K_{m}$ 값을 보였다.

• Environmental Analysis Health and Toxicology
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• 제16권2호
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• pp.97-102
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• 2001

FMOs (Flavin-containing monooxygenases, EC1.14.13.8)는 다양한 종류의 식품, 약물이나 기타 외래 유래물질 (xenobiotics)를 산화시키는 NADPH와 $O_2$ 의존성 약물대사효소이다. 현재까지 5종의 subfamily가 존재하는 것으로 보고되어 있으며 그 중 잘 알려진 FMO3는 대표적 인 subfamily로서 주로 간에 존재한다 사람FMO에 관한 연구는 최근 들어 활성화되기 시작했으며 질소, 황이나 인 등을 포함하는 친핵성 (nucleophilic) 화합물이 대표적인 기질로 보고되어 있다. 본 연구에서는 thiocarbamide를 포함하고 있는 화합물에 대한 사람의 FMO3의 기질 특이성을 알아보고자 하였다. 사람 FMO3를 baculovirus system을 이용하여 대량으로 발현시킨 후 그 microsomal FMO3를 분리하여 thiocholine assay를 시행하였다. 그 결과 methimazole, thiourea, and phenylthiourea는 낮은 $K_{m}$ (4-10$\mu$M)간을 갖는 반면, 이보다 기질의 크기가 큰 1 ,3-diphenylthiourea, 1, 3-bis (3, 4-dichlorophenyl)-2-thiourea, 1, 1-dibenzyl-3-phenol-2-thiourea에서는 효소활성이 나타나지 않았다. 이는 사람 fM01과 비교하여 볼 때 큰 차이는 없었으며, 다른 pig, guinea pig, rat, rabbit에서 보다 받아들일 수 있는 기질의 크기가 더 제한적임을 알 수 있었다.

• The Korean Journal of Physiology and Pharmacology
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• 제3권2호
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• pp.207-213
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• 1999

To examine individual variation in drug metabolism catalyzed by flavin-containing monooxygenase (FMO), 179 Korean volunteers' urinary molar concentration ratio of theobromine (TB) and caffeine (CA) was determined. Their urine was collected for 1 hr (between 4 and 5 hrs) after they drank a cup of coffee containing 115 mg CA and analyzed by an HPLC system. The lowest TB/CA ratio obtained was 0.40, the highest ratio was 15.17 (38-fold difference), and the median ratio for all subjects was 1.87. The mean was 2.66 with 2.36 S.D.. In 134 nonsmokers, the mean ratio was $2.35{\pm}1.93,$ that of 51 males was $2.30{\pm}2.26$ and 83 females was $2.37{\pm}1.85,$ respectively. There was no significant gender difference in the obtained TB/CA ratio (Mann-Whitney test; p=0.518). There were no smokers among the 83 female volunteers. In the remaining 96 male subjects, the ratio obtained in 51 nonsmokers was $2.30{\pm}2.06$ and that of 45 smokers was $3.62{\pm}3.19.$ This indicated that the TB/CA ratio was increased significantly in smokers (p=0.007). However, when the TB/CA ratios (FMO activity) obtained in all 179 Korean volunteers are compared with the urinary concentration ratios of paraxanthine (PX) plus 1,7-dimethylurate (17U) to CA (CYP1A2 activity), there was a weak but significant correlation (Pearson's correlation coefficient test; $r^2=0.28,$ p＜0.0001). This indicates that, although the urinary TB/CA ratio mostly represents FMO activity, minor contribution by CYP1A2 activity cannot be ignored. In conclusion, the FMO activity measured by taking the urinary TB/CA ratio from normal healthy Korean volunteers shows marked individual variations without significant gender differences and the increased TB/CA ratio observed in cigarette smokers may have been caused by the increased CYP1A2 activity.

• 한국식품위생안전성학회지
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• 제14권4호
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• pp.415-421
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• 1999

The flavin-containing monooxygenases (FMOs) (EC 1.14. 13.8) are NADPH-dependent flavoenzymes that catalyze oxidation of soft nucleophilic heteroatom centers in a range of structurally diverse compounds including foods, drugs, pesticides, and other xenobiotics. In humans, FMOl appears to be the predominant form expressed in human fetal liver. cDNA-expressed human FMO and human liver microsomal FMO have been observed to N- and S-oxy-genate nucleophilic nitrogen- and sulfur-containing drugs and chemicals, respectively. In the present study, FMOl can be expressed in the baculovirus expression vector system at level of 2.68 nmol FMOl/mg of membrane protein. This isoform was examined for its capacity to metabolize methimazole to its S-oxide using thiocholine assay. Kinetic studies of its S-oxide by recombinant human FMO1 result in Km of 7.66 $\mu$M and Vmax of 17.79 nmol/min/mg protein.

• 한국연초학회지
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• 제20권1호
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• pp.99-107
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• 1998

Numerous studies have demonstrated a negative association between cigarette smoking and Parkinson's disease. The present study was undertaken to investigate whether chronic exposure of mice to cigarette smoke a(footed the metabolism of 1-methyl-1113,6-tetrahydro-pyridine (MPTP) by cytochrome P4SO (P-450) or flavin-containing monooxygenase (FMO) in the hepatic microsomes of C57BL6/J mice. Adult male C57BL6/J mice were exposed to mainstream smoke generated from 15 cigarettes for 10 min a day and 5 day per week for 6 weeks. MPTP (10 mg/kg body weight) was administered to mice by subcutaneous injection for 6 consecutive days. Microsolnal P-450 content was increased by MPTP, smoke exposure, or both, but NADPH cytochrome P-450 reductase activity was rather decreased by the same treatments. The activities of benzo(a)pyrene hydroxylase, 7-ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase were significantly increased by the exposure of cigarette smoke, but were not or little affected by MPTP treatment. Benzphetamine N-demethylase activity was not affected either by MPTP treatment or by cigarette smoke exposure, but it was significantly increased by the combined MPTP treatment with cigarette smoke exposure, showing their synergic effect for the induction of the enzyme activity. Interestingly, in vitro studies of hepatic FMO and P-450 system both O-oxygenation and N-demethylation of MPTP were increased in the smoke-exposed or in the MPTP-treated mice. These results suggest that the enhancement in the N-demethylation as well as O-deethylation of P-450 system and in the N-oxygenation of FMO activity by cigarette smoke exposure in mouse liver may contribute to attenuating the neurotoxic effects of MPTP on the nigrostriatal dopaminergic neurons.

• Archives of Pharmacal Research
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• 제27권5호
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• pp.547-553
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• 2004

The pyrrolizidine alkaloids (PAs), contained in a number of traditional remedies in Africa and Asia, show wide variations in metabolism between animal species but little work has been done to investigate differences between animal strains. The metabolism of the PA senecionine (SN) in Fischer 344 (F344) rats has been studied in order to compare to that found in the previously investigated Sprague-Dawley (SO) rats (Drug Metab. Dispos. 17: 387, 1989). There was no difference in the formation of ($\pm$) 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP, bioactivation) by hepatic microsomes from either sex of SO and F344 rats. However, hepatic microsomes from male and female F344 rats had greater activity in the Noxidation (detoxication) of SN by 88% and 180%, respectively, when compared to that of male and female SD rats. Experiments conducted at various pH showed an optimum pH of 8.5, the optimal pH for flavin-containing monooxygenase (FMO), for SN N-oxidation by hepatic microsomes from F344 females. In F344 males, however, a bimodal pattern was obtained with activity peaks at pH 7.6 and 8.5 reflecting the possible involvement of both cytochrome P450 (CYP) and FMO. Use of specific inhibitors (SKF525A, 1-benzylimidazole and methimazole) showed that the N-oxide of SN was primarily produced by FMO in both sexes of F344 rats. In contrast, SN N-oxide formation is known to be catalyzed mainly by CYP2C11 rather than FMO in SD rats. This study, therefore, demonstrated that there were substantial differences in the formation of SN N-oxide by hepatic microsomes from F344 and SD rats and that this detoxification is catalyzed primarily by two different enzymes in the two rat strains. These findings suggest that significant variations in PA biotransformation can exist between different animal strains.