Activities of daily living and oral hygiene status of older Korean patients in a long-term care facility.

OBJECTIVES: This study investigated the relationship between oral hygiene conditions, activities of daily living (ADL) and cognitive ability in older Korean patients in long-term care facilities. METHODS: Ninety older persons (65+) were randomly sampled from a possible 112 residents in a single facility. They participated in a 2-month-long survey. The Korean Modified Barthel Index was used to measure the ADL, and cognitive ability was measured using the Mini-Mental State Examination, Korean version. Oral hygiene status was measured using the Simplified Oral Hygiene Index and the Tongue Coating Index (TCI). RESULTS: Older participants with complete dependence manifested significantly poorer oral hygiene (P < 0.05). Scores on the TCI were significantly higher in participants who were dentulous with partial dependence (P < 0.05). A multiple regression analysis showed that dependence and being dentulous significantly predicted poorer oral hygiene (P < 0.05). CONCLUSIONS: This study suggests that older participants with complete dependence had poor oral hygiene on tooth surfaces, while participants with partial dependence had poor tongue hygiene. In addition, dentulous older participants had poorer tongue hygiene than edentulous ones. This indicates the need to assess tooth status and provide oral care services via ADL in long-term care facilities.

Gender differences in chewing discomfort in older South Koreans.

AIMS: The purpose of this study was to identify gender differences in chewing discomfort among elderly Koreans. METHODS: This study used data from 56 616 (weighted sample: 5 638 394) subjects aged over 65 years who participated in the 2011 Community Health Survey in Korea. Of them, 23 059 (weighted sample: 2 368 200, 42.0%) were men and 33 357 (weighted sample: 3 270 194, 58.0%) were women. Data were analysed using chi-square tests and hierarchical logistic regression analyses, with SPSS 20.0. Chewing discomfort was set as the dependent variable, and independent variables were divided into socio-economic factors (place of residence, age, education, monthly household income, basic living security stipend, private insurance, economic activity, living arrangements), general health factors (hypertension, diabetes) and oral health factors (tooth defects, denture use, subjective periodontal health status). RESULTS: A greater proportion of women (50.2%) than men (42.6%) exhibited chewing discomfort (P < 0.001). In men, place of residence, monthly household income, private health insurance, tooth defects and periodontal health were associated with chewing discomfort (P < 0.05). In women, age, education level, basic living security stipend and denture use were associated with chewing discomfort (P < 0.05). CONCLUSIONS: Elderly Korean women experience more severe chewing discomfort than their male counterparts. The factors associated with chewing also differ by gender.

Community periodontal treatment needs in South Korea.

OBJECTIVES: This study aimed to assess the relationship between socio-economic factors and community periodontal treatment needs in Korea. METHODS: Data were obtained from the year 2009 Korean National Health and Nutrition Examination Survey. Our analysis included 7510 survey participants over the age of 19 years. To assess the relationship between socio-economic factors and the need for periodontal scaling, we performed multivariate logistic regression analyses for data with a complex sampling structure. PASW statistics 19.0 (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analyses, and the results were expressed as odds ratios (OR) with corresponding 95% confidence intervals (CIs). RESULTS: A very high percentage of Korean adults required periodontal scaling (71.5%). After adjusting for sex, age, and socio-economic factors, the need for periodontal scaling was associated with low levels of education (OR: 1.41, 95% CI: 1.03-1.93), low incomes (OR: 1.27, 95% CI: 1.01-1.60), employment as a service and sales worker (OR: 1.39, 95% CI: 1.10-1.77), and employment as a manual worker (OR: 1.31, 95% CI: 1.02-1.69). CONCLUSIONS: In South Korea, the need for periodontal scaling was associated with socio-economic factors, such as low levels of education, low incomes, employment as a service and sales worker and employment as a manual worker. Consequently, clinical and community dental hygienists should consider adults with these risk factors as belonging to high-priority subgroups to whom they should respond first.

Reduction in dental plaque in patients with mental disorders through the dental hygiene care programme.

OBJECTIVE: To develop a dental hygiene care programme based on the specific needs of patients with mental disorders and to suggest practical guidelines to improve the oral health care of these patients. METHODS: A total of 73 patients with mental illness participated in the study. The patients were randomly classified into three groups and followed over 12 weeks at 4-week intervals. A newly designed dental hygiene care programme using flash-based video, brochures and a toothpick method was implemented by five dental hygienists. Plaque index, stimulated saliva, subjective oral dryness and dental caries activity were analysed as outcome variables. RESULTS: Results showed that the dental plaque index significantly decreased after each session (P < 0.0001) in all three groups, and significant differences were found between groups (P = 0.036). Patients' oral dryness decreased significantly, but stimulated saliva and dental caries activity did not improve. CONCLUSION: The results of this study suggest that the dental hygiene care programme, which made use of a short, 10-min flash-based video and brochures every 4 weeks, was effective in reducing the dental plaque index of patients with mental disorders.

The effect of orally administered epigallocatechin-3-gallate on ligature-induced periodontitis in rats.

BACKGROUND AND OBJECTIVE: Epigallocatechin-3-gallate (EGCG) is known for its beneficial properties, including anti-inflammatory and anti-oxidative activities. Recently, reports have suggested that EGCG plays a pivotal role in regulating cytokine expression and osteoclastic activity. In the present study, we investigated whether orally administered EGCG has a therapeutic effect on ligature-induced periodontitis. MATERIALS AND METHODS: Forty-eight Sprague-Dawley rats were treated with EGCG or phosphate-buffered saline. Periodontitis was induced by tying a ligature for 7 d. After removing ligation, EGCG (200 mg/kg) or phosphate-buffered saline was administered via oral gavage on a daily basis. Rats were killed after 1, 2 and 4 wk of administration. Histologic and histomorphometric analyses, tartrate resistant acid phosphatase staining and immunohistochemistry were carried out. RESULTS: In the control group, bone loss did not recover even after the causative factor of periodontitis was eliminated. On the other hand, distance from cemento-enamel junction to alveolar bone crest, long junctional epithelium and collagen destruction were reduced in the EGCG group. Decreased interleukin (IL)-6 expression was shown from the early stage of EGCG administration, followed by reduced tumor necrosis factor (TNF) expression at week 4 EGCG group. The CT area showed a higher decrease of IL-6 expression between the control and EGCG group than alveolar bone area. Downregulation of TNF and IL-6 expression led to a decrease in osteoclast number and activity, which resulted in reduced bone loss. CONCLUSIONS: Systemic administration of EGCG could have a therapeutic effect on damaged periodontal tissue. Inhibited cytokine expression, including TNF and IL-6 is responsible for the reduction in osteoclast formation, osteoclastic activity and collagen destruction.

Intramuscular immunoglobulin for recalcitrant suppurative diseases of the skin: a retrospective review of 63 cases.

BACKGROUND: Intramuscular human immunoglobulin (HIG) may provide a therapeutic option as an independent or combined treatment for recalcitrant suppurative skin diseases such as hidradenitis suppurativa, folliculitis decalvans, or chronic recurrent furunculosis or folliculitis. OBJECTIVES: To define the efficacy and safety of intramuscular HIG for chronic and recalcitrant suppurative skin diseases. METHODS: Patients who had received HIG for hidradenitis suppurativa, folliculitis decalvans, furunculosis or folliculitis at Severance Hospital, Seoul, Korea, between January 2000 and May 2005 were identified from medical/pharmacy records. All records were analysed retrospectively. RESULTS: Sixty-three patients were identified. After treatment, 37 patients (59%) showed overall improvement and were rated as having an 'excellent response' or 'good response' by the attending physician. No improvement or worsening was seen in only three patients (5%). A period without new lesions (PWNL) was achieved in 46 patients (73%). The number of times HIG was administered to achieve PWNL ranged from 1 to 12 (mean +/- SD 2.15 +/- 1.69). There was no significant difference in the rating score between the independent intramuscular HIG and the combined treatment groups. Pain at the injection site was the major side-effect, which led to the discontinuation of treatment in five patients. No other significant systemic side-effects were observed. CONCLUSIONS: Our results demonstrate that intramuscular HIG may be used for the treatment of recalcitrant suppurative skin diseases as an independent or combined treatment.

Comparative in vitro metabolism of benzo[a]pyrene by recombinant zebrafish CYP1A and liver microsomes from beta-naphthoflavone-treated rainbow trout.

The zebrafish (Danio rerio) is a sensitive non-mammalian model used for studying polycyclic aromatic hydrocarbon (PAH)-induced chemical carcinogenesis. The susceptibility of zebrafish to PAH-induced carcinogenesis may be related to the ability of the zebrafish P450s to bioactivate these procarcinogens. As a part of our overall effort to identify the various P450 enzymes that are involved in the activation and detoxification of PAHs in zebrafish, therefore, we have examined the ability of recombinant zebrafish CYP1A (zCYP1A) expressed in yeast to metabolize BaP in vitro. Comparison studies also were conducted with liver microsomes from beta-naphthoflavone (BNF)-treated rainbow trout (Oncorhynchus mykiss). Results demonstrated that the trout liver microsomes were almost twice as active as zCYP1A in oxidizing BaP, with Vmax values of 1.7 and 0.94 nmol/min/nmol P450 for trout and zebrafish preparations, respectively. Like trout CYP1A1, cDNA-expressed zCYP1A was found to oxidize BaP to phenols, quinones and diols (BaP-7,8-diol and BaP-9,10-diol) in the presence of exogenous human microsomal epoxide hydrolase (hEH). BaP-7,8-diol is the precursor of the ultimate carcinogen, BaP-7,8-diol-9,10-epoxide (BaPDE). The ability of zCYP1A to bioactivate BaP was confirmed by the formation of DNA adducts when calf thymus DNA was added to the incubation mixture. BaP-DNA binding was enhanced by the addition of hEH to the incubation mixture. HPLC analysis of the [33P]-postlabeled DNA adducts showed the formation of at least four adducts mediated by both zCYP1A and trout liver microsomes, and one of these adducts co-migrated with BaPDE-dG in HPLC analysis. The addition of hEH to the incubation mixture decreased the formation of BaPDE-dG by zCYP1A and by trout liver microsomes while increasing the formation of an unidentified DNA adduct in the case of zCYP1A. zCYP1A also mediated the binding of BaP to protein, providing further evidence that this enzyme is capable of oxidizing BaP to reactive metabolites that bind to macromolecules. It thus appears that zCYP1A may play an important role in BaP-induced carcinogenesis in the zebrafish model by catalyzing the sequential formation of the ultimate diol epoxide carcinogenic metabolite of BaP.

CYP2K6 from zebrafish (Danio rerio): cloning, mapping, developmental/tissue expression, and aflatoxin B1 activation by baculovirus expressed enzyme.

A full-length zebrafish (Danio rerio) cytochrome P450 (CYP) 2K6 cDNA, was obtained (GenBank accession No. AF283813) through polymerase chain reaction cloning using degenerated primers based on a consensus CYP2 sequence and the heme-binding domain. This first CYP2K family member cloned from zebrafish had 1861 bp which contained 27 bp of 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 1518 bp, and a 300 bp 3'-UTR with a poly A tail. The deduced 506 amino acid sequence of CYP2K6 had 63%, 62% and 59% identity with rainbow trout CYP2K1, CYP2K4 and CYP2K3, respectively; and 45%, 42%, and 42% identity with rabbit CYP2C1, human CYP2C19 and mouse CYP2C39, respectively. CYP2K6 mapped to 107.49cR on LG3 using the LN54 radiation hybrid panel. Its mRNA was detected at 5 days post-fertilization and in the adult liver and ovary among nine tissues examined. The ORF, including the 27 bp of the 5'-UTR, was cloned into pFastBac donor vector and then transferred into the baculovirus genome (bacmid DNA) in DH10Bac competent cells. The recombinant bacmid DNA was used to infect Spodoptera frugiperda insect cells to express the CYP2K6 protein (Bv-2K6). As its ortholog, rainbow trout Bv-2K1 [Yang, Y.H., Miranda, C.L., Henderson, M.C., Wang-Buhler, J.-L., Buhler, D.R., 2000. Heterologous expression of CYP2K1 and identification of the expressed protein (Bv-2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase. Drug Metab. Disp. 28,1279-83.], Bv-2K6 also catalyzed the conversion of aflatoxin B1 (AFB1) to its exo-8,9-epoxide as assessed by the trapping of a glutathione (GSH) adduct in the presence of a specific mouse alpha class glutathione S-transferase. The identity of the AFB1-GSH adduct was verified by liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry-mass spectrometry (MS-MS) analysis. Although rainbow trout Bv-2K1 was capable of oxidizing lauric acid, zebrafish Bv-2K6 protein showed no activity against this substrate.

Risperidone metabolism in relation to CYP2D6*10 allele in Korean schizophrenic patients.

OBJECTIVE: Risperidone is known to be biotransformed to its active metabolite, 9-hydroxyrisperidone, by the polymorphic CYP2D6 in Caucasians. This study aimed to investigate the relationship between the CYP2D6*10 allele and the plasma levels of risperidone and 9-hydroxyrisperidone in Korean schizophrenic patients. METHODS: Eighty-two Korean schizophrenic patients in monotherapy with oral doses of risperidone from 1 mg/day to 8 mg/day (mean +/- SD 4.3 +/- 1.9, median 4) participated in this study. Plasma concentrations of risperidone and 9-hydroxyrisperidone were analyzed using high-performance liquid chromatography. The CYP2D6*10 allele, which contains C188T mutation in exon 1, was identified using allele-specific polymerase chain reaction amplification. RESULTS: Seventeen of 82 patients were homozygous for CYP2D6*1, 22 for *10, while the remaining 43 patients were heterozygous for these alleles. The plasma levels of risperidone and 9-hydroxyrisperidone ranged from 1.0 nM to 168 nM and 6.2 nM to 235 nM, respectively. The median concentrations/dose (C/Ds) (range) of risperidone in CYP2D6*1/*1, *1/*10, and *10/*10 groups were 1.7 (0.2-7.9), 2.6 (0.3-27.1), and 6.7 nM/mg (2.4-21.0), respectively. There was a statistically significant difference among the three genotypes (Kruskal-Wallis test, P<0.001). For 9-hydroxyriperidone, the corresponding median C/Ds were 13.1 (3.3-25.4), 11.9 (4.2-30.8), and 13.6 nM/mg (6.5-52.8), respectively, with no significant difference between the genotypes (P=0.54). The medians of the ratios between risperidone and 9-hydroxyrisperidone concentrations were 0.13 (0.01-0.93), 0.28 (0.01-2.77), and 0.46 nM/mg (0.05-1.28) in *1/*1, *1/*10, and *10/*10 genotypes, respectively, and they were significantly different (P=0.004). The active moieties (sum of the C/Ds of risperidone and 9-hydroxyrisperidone) were not significantly different between the genotypes (P=0.063). CONCLUSION: In Korean schizophrenic patients, the metabolism of risperidone is dependent on CYP2D6, and the CYP2D6*10 allele is important for the regulation of the activity of this enzyme. There were no significant differences in the plasma concentration of parent drug plus its active metabolite between the genotypes. This suggests that the clinical significance of this polymorphism is limited. Our study confirms previous studies on risperidone metabolism in Caucasians.

Oxidation of ranitidine by isozymes of flavin-containing monooxygenase and cytochrome P450.

Rat and human liver microsomes oxidized ranitidine to its N-oxide (66-76%) and S-oxide (13-18%) and desmethylranitidine (12-16%). N- and S-oxidations of ranitidine were inhibited by metimazole [flavin-containing monooxygenase (FMO) inhibitor] to 96-97% and 71-85%, respectively, and desmethylation of ranitidine was inhibited by SKF525A [cytochrome P450 (CYP) inhibitor] by 71-95%. Recombinant FMO isozymes like FMO1, FMO2, FMO3 and FMO5 produced 39, 79, 2180 and 4 ranitinine N-oxide and 45, 0, 580 and 280 ranitinine S-oxide pmol x min(-1) x nmol(-1) FMO, respectively. Desmethyranitinine was not produced by recombinant FMOs. Production of desmethylranitidine by rat and human liver microsomes was inhibited by tranylcypromine, a-naphthoflavon and quinidine, which are known to inhibit CYP2C19, 1A2 and 2D6, repectively. FMO3, the major form in adult liver, produced both ranitidine N- and S-oxides at a 4 to 1 ratio. FMO1, expressed primarily in human kidney, was 55- and 13-fold less efficient than the hepatic FMO3 in producing ranitidine N- and S-oxides, respectively. FMO2 and FMO5, although expressed slightly in human liver, kidney and lung, were not efficient producers of ranitidine N- and S-oxides. Thus, urinary contents of ranitidine N-oxide can be used as the in vivo probe to determine the hepatic FMO3 activity.

Differential and constitutive expression of neuronal, inducible, and endothelial nitric oxide synthase mRNAs and proteins in pathologically normal human tissues.

Nitric oxide (NO) is produced by NO synthases (nNOS, iNOS, and eNOS) expressed in various human tissues and depending on the amount of NO produced in each tissue, the physiological function of NO is determined. However, due to the difficulty in obtaining normal human tissues, little is known about the basal levels of each of the three NOS mRNAsand proteins expressed constitutively in various human tissues. Results of the present study indicate that the basal levels of each of the three NOS mRNAs and proteins expressed in various regions of brain and peripheral tissues are different both in their sizes and in their contents. In Northern blot analysis, two different-sized mRNAs were found for each NOS isozymes: for the nNOS, approximately 12 and <12 kb mRNAs; for the iNOS, 4.2 and 4.5 kb mRNAs; for the eNOS, 4.2 and 4.4 kb mRNAs. In the Western blot, several different-sized NOS proteins were detected ( approximately 160, approximately 140, and approximately 130 kDa for nNOS; approximately 130 kDa for iNOS and eNOS) with tissue-specific expression patterns. These differential expression patterns of NOS mRNAs and proteins were caused by alternative splicing in the open-reading frame, and 5'- and/or 3'-untranslated regions of NOS mRNAs. These results suggest that regulation for differential expression of the three NOS genes in various human tissues may occur by alternative splicing of the NOS mRNAs in tissue-specific patterns.

Phenotypes of flavin-containing monooxygenase activity determined by ranitidine N-oxidation are positively correlated with genotypes of linked FM03 gene mutations in a Korean population.

A non-invasive urine analysis method to determine the in-vivo flavin-containing mono-oxygenase (FMO) activity catalysing N-oxidation of ranitidine (RA) was developed and used to phenotype a Korean population. FMO activity was assessed by the molar concentration ratio of RA and RANO in the bulked 8 h urine. This method was used to determine the FMO phenotypes of 210 Korean volunteers (173 men and 37 women, 110 nonsmokers and 100 smokers). Urinary RA/RANO ratio, representing the metabolic ratio and the reciprocal index of FMO activity, ranged from 5.67-27.20 (4.8-fold difference) and was not different between men and women (P = 0.76) or between smokers and nonsmokers (P = 0.50). The frequencies of RA/RANO ratios were distributed in a trimodal fashion. Among the 210 Korean subjects, 93 (44.3%) were fast metabolizers, 104 (49.5%) were intermediate metabolizers and 13 (6.2%) were slow metabolizers. Subsequently, the relationship between the ranitidine N-oxidation phenotypes and FMO3 genotypes, determined by the presence of two previously identified mutant alleles (Glu158Lys: FMO3/Lys158 and Glu308Gly: FMO3/Gly308 alleles) commonly found in our Korean population was examined. The results showed that subjects who were homozygous and heterozygous for either one or both of the FMO3/Lys158 and FMO3/Gly308 mutant alleles had significantly lower in-vivo FMO activities than those with homozygous wild-type alleles (FMO3/Glu158 and FMO3/Glu308) (P < 0.001, Mann-Whitney U-test). Furthermore, the FMO activities of subjects with either FMO3/Lys158 or FMO3/Gly308 mutant alleles were almost identical to those having both FMO3 mutant alleles (FMO3/Lys158 and FMO3/Gly308). These two mutant alleles located, respectively, at exons 4 and 7 in the FMO3 gene appeared to be strongly linked by cis-configuration in Koreans. Therefore, we concluded that presence of FMO3/Lys158 and FMO3/Gly308 mutant alleles in FMO3 gene is responsible for the low ranitidine N-oxidation (FMO3 activity) in our Korean population.

Effect of age and smoking on in vivo CYP1A2, flavin-containing monooxygenase, and xanthine oxidase activities in Koreans: determination by caffeine metabolism.

OBJECTIVES: To assess the effect of gender, age, and smoking habits on the in vivo activities of CYP1A2, flavin-containing monooxygenase (FMO), and xanthine oxidase in Korean subjects. METHODS: One hundred thirty-three age- and gender-matched healthy Korean volunteers (age range, 21 to 78 years; mean age, 35.3 +/- 16.6 years) with and without smoking habits participated. After drinking a cup of coffee (200 mL) that contained 110 mg caffeine, a 1-hour urine sample (between 4 and 5 hours) was collected and caffeine metabolites were analyzed by HPLC. RESULTS: There were marked individual variations in CYP1A2 [(1,7-dimethylurate + paraxanthine)/caffeine], FMO (theobromine/caffeine), and xanthine oxidase (1-methylurate/1-methylxanthine) activities (14-, 42-, and 9-fold, respectively). However, the mean values of these enzyme activities in the nonsmokers were not different between men and women. In the nonsmoking subjects in their 20s, the mean values of CYP1A2 and FMO activities (13.5 +/- 5.9 and 2.1 +/- 1.9, respectively) were higher than those (7.9 +/-1.8 and 0.95 +/- 0.22) of older decennial age groups. Xanthine oxidase activities were the same for all age groups (subjects in their 20s through their 70s). CYP1A2 activity of the smokers (20.0 +/- 9.6) was higher than that of the nonsmokers (10.8 +/- 5.8; P < .001). Similarly, the FMO activity in smokers (3.4 +/- 2.7) was higher than that of the nonsmokers (1.8 +/- 1.7; P < .001). The xanthine oxidase activity (1.3 +/- 0.5) was not increased in smokers (1.4 +/- 0.5; P = .46). CONCLUSIONS: Results of this caffeine metabolism study conducted with age- and gender-matched healthy Korean volunteers with and without smoking habits provided the baseline and the widely varying interindividual activities of CYP1A2, FMO, and xanthine oxidase in a Korean population. The results also suggested that drugs metabolized by CYP1A2 and FMO may require individualized dose adjustment according to the age and smoking habits of the subjects.

Co-administration of toluene and xylene antagonized the testicular toxicity but not the hematopoietic toxicity caused by ethylene glycol monoethyl ether in Sprague-Dawley rats.

Occupational painters are exposed to ethylene glycol monoethyl ether (EGEE), a widely used emulsifying solvent known to cause testicular degeneration and bone marrow depression, together with toluene (TOL) and xylene (XYL) as a mixture. In the previous study (Chung et al., Tox. Lett. 104:143, 1999), testicular atrophy caused by EGEE (200 mg/kg) was shown to be antagonized by co-administration of TOL (250 mg/kg) and XYL (500 mg/kg). This study was conducted to provide histological support for the previously observed antagonistic protective effect of TOL + XYL on EGEE inducible testicular toxicity and to determine whether a similar antagonistic effect can be demonstrated against the EGEE derived hematopoietic toxicity. Compared to the extent of seminiferous tubule degeneration caused by EGEE (150 mg/kg, six times per week for 4 weeks), testes of rats given co-administration of TOL (250 mg/kg) + XYL (500 mg/kg) showed dramatically reduced tubular degeneration. Hyperplasia of Leydig cells in the interstitium was observed in both EGEE and EGEE + TOL + XYL-treated rats. Although a minimal dose of EGEE causing testicular atrophy was used, WBC and platelet counts were decreased significantly. In the TOL + XYL-treated control group, the WBC and platelet counts were not decreased. However, the bone marrow depression caused by EGEE was not reversed by the combined administration of TOL + XYL. In all experimental groups (EGEE alone, TOL + XYL, EGEE + TOL + XYL), plasma levels of creatinine and alkaline phosphatase were significantly decreased. In addition to the marked testicular atrophy, EGEE also decreased the weights of adrenal glands and epididymis. In conclusion, while the testicular degeneration caused by EGEE was antagonized by TOL + XYL, the EGEE derived hematopoietic suppression was not reversed.

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.

Phenotyping of flavin-containing monooxygenase using caffeine metabolism and genotyping of FMO3 gene in a Korean population.

Flavin-containing monooxygenase (FMO) activity was determined in 82 Korean volunteers by taking molar concentration ratio of theobromine and caffeine present in the 1 h urine (between 4 and 5 h) samples collected after administration of a cup of coffee containing 110 mg of caffeine. Among 82 volunteers, there were 19 women and 63 men (30 smokers and 52 non-smokers). Volunteers were divided into two groups comprising low (0.53-2.99) and high (3.18-11.95) FMO activities separated by an antimode of 3.18. Peripheral bloods were sampled from these volunteers and their genomic DNAs were amplified by polymerase chain reaction with oligonucleotides designed from intronic sequences of human FMO3 gene. Comparing nucleotide sequences of the amplified FMO3 gene originating from randomly selected individuals with low and high FMO activities, nine point mutations were identified in the open reading frame sequences. Among these nine mutations, three FMO3 mutant types (FMO3/Stop148, Lys158 and Gly308) were selected and correlated with FMO activities observed in our Korean population. A rare FMO3/Stop148 mutant allele originating from FMO3/Gly148 occurred by substitution of G442T in exon 4 and yielded a premature TGA stop codon. The stop codon was detected in one individual having the second lowest FMO activity and he had the mutation in heterozygous state. In a pedigree study, he was found to have inherited the mutation from his mother who also had a heterozygous stop codon and equally low FMO activity. In our volunteers, two other common mutations were detected in exons 4 and 7. The one in exon 4 resulted from a G472A change eliminating a HinfI restriction site and produced an amino acid substitution from Glu158 to Lys. The other mutation in exon 7 resulted from an A923G change generating a DraII restriction site and produced a non-conservative replacement of Glu308 to Gly. Based on the secondary structure maps of FMO3 enzyme proteins for these two mutant types, FMO3/Gly308 mutation transformed the helix structure into a sheet shape and indicated that dysfunctional FMO3 may be produced. FMO3/Lys158 mutation did not alter the secondary structure. Approximately 80% of volunteers with homozygous and/or heterozygous mutations on either one or two of these mutations had low FMO activities. Thus, individuals with these FMO3 gene mutations may have defective metabolic activity for many clinically used drugs and dietary plant alkaloids which are oxidized primarily by hepatic FMO3.

Decreased formation of ethoxyacetic acid from ethylene glycol monoethyl ether and reduced atrophy of testes in male rats upon combined administration with toluene and xylene.

Male painters are commonly exposed to ethylene glycol monoethyl ether (EGE), a well known reproductive toxic agent causing testicular atrophy, in the form of solvent mixture containing toluene (TOL) and xylene (XYL). This study was carried out to determine the effect of exposing male rats to solvent mixture containing TOL and XYL on the EGE (200 mg/kg) on testicular atrophy and production of toxic metabolite, ethoxyacetic acid (EAA) from EGE. Compared to the extent of testes atrophy observed upon EGE administration alone, the combined administration of EGE (200 mg/kg) with TOL (250 mg/kg) and XYL (500 mg/kg) for 4 weeks has reduced the extent of testes atrophy by 25%. The combined administration delayed the time for appearance of the highest plasma concentration (t(max)) of EAA from 3 to 6 h and also decreased the highest concentration (Cmax) as well as the total amount of plasma EAA (AUC(0-18 h)) by 45 and 29%, respectively. This explained the diminished testicular atrophy in male rats observed when EGE was administered in a solvent mixture containing TOL and XYL. This study suggested that testicular toxicity observed in male painters caused by EGE may be decreased when they are exposed to EGE in the form of solvent mixture containing TOL and XYL.

Involvement of CYP3A1, 2B1, and 2E1 in C-8 hydroxylation and CYP 1A2 and flavin-containing monooxygenase in N-demethylation of caffeine; identified by using inducer treated rat liver microsomes that are characterized with testosterone metabolic patterns.

Caffeine (CA) is oxidized by rat liver microsomal enzymes to theobromine (TB), paraxanthine (PX), and theophylline (TP) by N-demethylation and to trimethylurate (TMU) by C-8 hydroxylation, In order to identify the specific enzymes responsible for productions of these primary CA metabolites, liver microsomes enriched with various isoforms of cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) are prepared by pretreatment of rats with several inducers. The specific increases in various CYP or FMO activities are identified with the diagnostic testosterone metabolic patterns or the thiobenzamide S-oxidation assay. They are then employed to metabolize the CA. Liver microsomes isolated from rats pretreated with phenobarbital (PB-microsomes) did not have increased FMO activity but had increased activities for hydroxylating the testosterone at 6 beta-(CYP3A1), 16 beta-(CYP2B1), and 2 beta-(CYP3A1) positions. This PB-microsomes had increased activity for TMU production from CA (result of C-8 hydroxylation). Liver microsomes isolated from rats pretreated with acetone (AC-microsomes) had a normal level of FMO activity but had enhanced rates of 6 beta-(CYP3A1) and 2 beta-(CYP3A1) hydroxylations of testosterone. The AC-microsomes again had increased activity for production of TMU. Similarly, the liver microsomes isolated from rats pretreated with dexamethasone (DEX-microsomes) had a normal level of FMO activity but had enhanced rates of forming 6 beta-and 2 beta-hydroxytestosterone (Cyp3A1) as well as androstenedione (CYP3A1). The DEX-microsomes again had increased activity for production of TMU only. Liver microsomes isolated from rats pretreated with 3-methylcholanthrene (MC-microsomes), however, had increased FMO activity and also enhanced rates of forming the 7 alpha-(CYP1A1/2, and 2A1), 6 beta-(CYP3A1), and 2 beta-(CYP3A1) hydroxytestosterone. The MC-microsomes had increased activity for producing all of the four primary metabolites of CA, i.e. the N-demethylation metabolites like TB, PX. and TP, as well as the C-8 hydroxylation metabolite TMU. By the process of association of the obtained results, liver microsomes with increased contents of CYP2B1, 3A1, and 2E1 could catalyze the C-8 hydroxylation at an increased rate producing increased amount of TMU. Increased productions of CA N-demethylation metabolites (TB, PX, and TP) are, however, catalyzed by the increased activities of CYP1A2 and FMO which are associated uniquely with the MC-microsomes.

Oxidation of caffeine to theobromine and theophylline is catalyzed primarily by flavin-containing monooxygenase in liver microsomes.

Upon N-demethylation of caffeine (CA) by rat and human liver microsomes, theobromine (TB), paraxanthine (PX), and theophylline (TP) are produced. The optimal pHs for the formation of TB, PX, and TP from CA by rat liver microsomes are 7.4 (most), 8.2 (minor) and 8.6 (moderate). At pH 7.4, PX is the primary metabolite formed and makes up 48% of the CA metabolites generated. In the presence of SKF525A, an inhibitor of P450 (CYP), the rates of TB, PX and TP production are inhibited by 32%, 68% and 42%, respectively. Alternatively, in the presence of methimazole, an inhibitor of flavin-containing monooxygenase (FMO), the rates of TB, PX and TP production are inhibited by 66%, 48% and 73%, respectively. In the presence of both SKF525A and methimazole, they are inhibited by 95%, 84% and 94%, respectively. With human liver microsomes, the CA is metabolized faster but is inhibited more extensively either by SKF525A (PX production) or by methimazole (TB production). Alternatively, when CA is metabolized at pH 8.6, the optimal pH of FMO catalyzed reaction, the rates of TB and TP formation are increased but the rate of PX production is decreased. Furthermore, at pH 8.6 and in the presence of methimazole, the rates of TB and TP formation are decreased by 82% and 95%, respectively. These results indicate that the FMO is responsible primarily for productions of TB and TP and the CYP for PX.

Induction of flavin-containing monooxygenase (FMO1) by a polycyclic aromatic hydrocarbon, 3-methylcholanthrene, in rat liver.

Flavin-containing monooxygenase (FMO), known not to be induced by xenobiotics, has been induced by a polycyclic aromatic hydrocarbon, 3-methylcholanthrene (3MC). We have found a prominent augmentation of hepatic FMO1 both at transcription and translation levels by pretreatment of rats with 3MC. Liver tissues were used to study the inductive effect of 3MC on the FMO1 isoform, the major form present in rat liver. Evidence for significant induction of rat FMO1 was observed in mRNA production (3.5 fold) identified from reverse transcription-polymerase chain reaction (RT-PCR) results. Induction was also seen in the catalytic activity of the enzyme (2.9 fold) as measured by the thiobenzamide S-oxidation assay using induced rat liver microsomes. Our finding is the first report to indicate that hepatic FMO1 can be induced with a polycyclic aromatic hydrocarbon compound. FMO plays crucial roles in the oxidation of N- and S-containing drugs. If FMO is also inducible with other environmental polyaromatic hydrocarbon compounds in general, this finding will have important consequences in understanding the altered half-lives of many clinically useful drugs.