Iron deficiency anemia increases nitric oxide production in healthy adolescents.

To investigate the influence of iron deficiency and iron supplementation on nitric oxide (NO) production, we measured serum iron markers, serum nitrate and nitrite (NOx) concentrations, reticulocyte maturity index (RMI), and serum transferrin receptor (sTfR) levels in 369 females aged 14-19 years. RMI was analyzed by flow cytometry, sTfR concentrations were measured by enzyme immunoassay, and serum NOx levels were tested by the Griess reaction. NOx concentrations of the subjects in iron depletion phase were significantly higher than those of healthy controls. NOx concentrations increased gradually as iron deficiency progressed and were threefold higher than for the healthy controls, when the subjects attained a frank iron deficiency anemia. In particular, the NOx concentrations were 7.5-fold higher in the patients with severe iron deficiency anemia (Hb<80 g/l) than for the subjects with high hemoglobin value (Hb>or=140 g/l). The increased NOx concentrations (132.6+/-42.1 microM) observed in the group with severe anemia decreased significantly (46.3+/-15.8 microM) after hemoglobin levels were normalized by iron supplementation ( P<0.01). NOx concentrations correlated inversely with hemoglobin levels ( r(2)=0.202, P<0.01), but correlated positively with the sTfR concentrations ( r(2)=0.322, P<0.01) and the RMI ( r(2)=0.369, P<0.01). In conclusion, iron deficiency anemia increases NO production, and elevated NOx concentrations in iron deficiency anemia return to normal with iron supplementation.

Cytochromes P450 and flavin monooxygenases--targets and sources of nitric oxide.

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 01 meeting in Orlando, FL. The presentations addressed the mechanisms of inhibition and regulation of cytochrome P450 and flavin monooxygenase enzymes by nitric oxide. They also highlighted the consequences of these effects on metabolism of drugs and volatile amines as well as on important physiological parameters, such as control of blood pressure, renal ion transport, and steroidogenesis. This is achieved via regulation of P450-dependent prostacyclin, hydroxyeicosatetraenoic acid, and epoxyeicosatrienoic acid formation. Conversely, the mechanisms and relative importance of nitric oxide synthases and P450 enzymes in NO production from endogenous and synthetic substrates were also addressed.

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.

Ethnic differences in human flavin-containing monooxygenase 2 (FMO2) polymorphisms: detection of expressed protein in African-Americans.

The flavin-containing monooxygenases (FMOs) are a family of xenobiotic-metabolizing enzymes that are expressed in a species- and tissue-specific manner. FMO2 expression has been observed in pulmonary tissue from several species, but not human. Two human FMO2 point mutations have been reported: a cytosine to thymidine transition at position 1414 resulting in a premature stop codon and a thymidine insertion at position 1589 resulting in a frameshift. To define the frequency of these sequence variations and explore their significance, unrelated African-American, Caucasian, and Korean individuals were genotyped. In the African-American population tested (n = 180), the 1414C allele occurred at a 13% frequency; however, all of the tested Caucasians (n = 52) and Koreans (n = 100) were homozygous for the 1414T allele. The T1589 allele occurred at frequencies of 6.9 and 13.0% in African-Americans (n = 175) and Caucasians (n = 23), respectively, and appears to segregate with the 1414T allele. Thus, it would have no further impact on FMO2 activity. Western blot analysis of pulmonary microsomes failed to detect immunoreactive protein in 1414T homozygotes. A heterozygotic individual did exhibit a single band of the expected size, but no detectable FMO activity in the corresponding lung microsomes. Sequence analysis, however, was consistent with the 1414C allele encoding an active FMO2 enzyme. FMO2 mRNA expression was observed in most individuals, but failed to correlate with genotype or protein expression. In summary, functional FMO2 is expressed in only a small percentage of the overall population. However, in certain ethnic groups, active pulmonary FMO2 enzyme will be present in a significant number of individuals.

Relationship of paroxetine disposition to metoprolol metabolic ratio and CYP2D6*10 genotype of Korean subjects.

OBJECTIVE: To evaluate the relationship between the metabolic ratio (MR) of metoprolol, CYP2D6*10B genotype, and the disposition of paroxetine in Korean subjects. METHODS: A single 40-mg dose of paroxetine was administered orally to one poor metabolizer and 15 healthy subjects recruited from 223 Korean extensive metabolizers whose phenotypes were predetermined by use of the metoprolol MR. Genotypes were determined by allele-specific polymerase chain reaction and the GeneChip microarray technique. Pharmacokinetic parameters were estimated from plasma concentrations of paroxetine for more than 240 hours after the oral dose. RESULTS: The oral clearance and area under the plasma concentration versus time curve (AUC) of paroxetine were best described by a nonlinear relationship with metoprolol MR at correlation coefficients of 0.82 and 0.91, respectively (P < .05). Nine extensive metabolizer who were either homozygous or heterozygous for CYP2D6*10B had significantly lower oral clearance values of paroxetine than six extensive metabolizers with CYP2D6*1/*1. The AUC of paroxetine in subjects who were homozygous for CYP2D6*10B (666.4 +/- 169.4 ng/mL x h) was significantly greater than that of subjects who were homozygous for the wild type (194.5 +/- 55.9 ng/mL x h). Unexpectedly, the average AUC of subjects who were heterozygous for CYP2D6*10B was greater with wide variation (789.8 +/- 816.9 ng/mL x h) than that of subjects who were homozygous CYP2D6*10B/*10B mainly because of two atypical subjects whose metoprolol MR was not associated with the CYP2D6*10B genotype and who showed greater AUC and lower oral clearance than subjects with homozygous CYP2D6*10B. CONCLUSIONS: The CYP2D6 activity measured by metoprolol MR was a strong predictor of paroxetine disposition in Korean extensive metabolizers. In general, the extensive metabolizers with the CYP2D6*10B allele seemed to have higher plasma concentrations of paroxetine than extensive metabolizers with the wild-type CYP2D6 genotype. However, quantitative prediction of paroxetine disposition from the CYP2D6*10B genotype alone was not perfect because several Korean extensive metabolizers had metoprolol MRs that were not associated with the genotype.

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.

Protective effect of ethanol against acetaminophen-induced hepatotoxicity in mice: role of NADH:quinone reductase.

The role of NAD(P)H:quinone reductase (QR; EC 1.6.99.2) in the alcohol-derived protective effect against hepatotoxicity caused by acetaminophen (APAP) was studied. In mice pretreated with dicoumarol (30 mg/kg), an inhibitor of QR, hepatic necrosis caused by APAP (400 mg/kg) was potentiated. Hepatocellular injuries induced by APAP, as assessed by liver histology, serum aminotransferase activities, hepatic glutathione (reduced and oxidized) contents, and liver microsomal aminopyrine N-demethylase activities, all were potentiated by pretreatment of mice with dicoumarol. Even in mice given APAP and ethanol (4 g/kg), in which APAP-inducible hepatic necrosis was abolished, the dicoumarol pretreatment again produced moderate hepatotoxicity and reversed the protective effect of ethanol. In mice pretreated with dicoumarol and ethanol, levels of APAP in blood and bile fluid between 90 and 240 min were higher than those in mice given ethanol. However, the biliary contents of sulfate and glucuronide conjugates of APAP were much lower than those in the ethanol group, particularly at early time points. In contrast, the biliary level of APAP-cysteine conjugate, which in the ethanol group was at its basal level, was increased maximally in the dicoumarol-pretreated mice. In the mice given dicoumarol and ethanol, the biliary APAP-cysteine conjugate level was increased moderately. These results suggest that ethanol inhibited not only the microsomal (CYP2E1 mediated) formation of a toxic quinone metabolite from APAP, but also accelerated the conversion of the toxic quinone metabolite produced back to APAP by stimulating cytoplasmic QR activity. In the presence of dicoumarol, however, QR activity was inhibited, and conversion of the toxic quinone metabolite back to APAP became inhibited and diminished the alcohol-dependent protective effect against APAP-induced hepatic injury.

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.

Silica increases cytosolic calcium and causes cell injury in renal cell lines.

The purpose of this study was to clarify the effect of silica-induced cytosolic free calcium mobilization and cell injury in immortalized cell lines from transgenic mice kidney harboring SV40 T-antigen gene. The proximal convoluted tubule (S1)- and the inner medullary collecting tubule (IMCT)-originated cell lines were used. Cytosolic free calcium concentration ([Ca2+]i) was measured employing Fura-2 fluorescence and cell injury was evaluated by a vital dye exclusion procedure. Silica increased [Ca2+]i in a concentration-dependent manner in S1 (60 micrograms/ml-600 micrograms/ml) and IMCT (6 micrograms/ml-600 micrograms/ml). Silica caused a biphasic increase in [Ca2+]i which was composed of an initial rapid rise and following sustained phase. Ca2+ removal from the medium resulted in abolishment of initial and sustained phase of silica (600 micrograms/ml)-induced [Ca2+]i in both cell lines. Silica-induced cell injury was increased in a dose-dependent manner. This silica-induced cell injury was attenuated by the pretreatment with EGTA (100 microM) and nifedipine (1 microM). Cellular ATP content ([ATP]i) by silica also decreased in a concentration-dependent manner. The relationship between [Ca2+]i and [ATP]i showed that [ATP]i depletion caused [Ca2+]i to rise. This study suggests that 1) the elevation of [Ca2+]i caused by silica is due mainly to influx through plasma membrane Ca2+ channel and non specific membrane damage (at high concentration) and 2) nephrotoxicity of silica shows site-specificity within the kidney.

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.

CYP2C19 genotype and phenotype determined by omeprazole in a Korean population.

Omeprazole (20 mg orally) was given to 103 healthy Korean subjects and blood was taken 3 h after administration. The plasma concentration ratio of omeprazole and hydroxyomeprazole, used as an index of CYP2C19 activity, was bimodally distributed. Thirteen subjects (12.6%) were identified as poor metabolizers (PMs) with an omeprazole hydroxylation ratio of 6.95 or higher. Among the 206 CYP2C19 alleles, CYP2C19*2 and CYP2C19*3 were found in 43 alleles (21%) and 24 alleles (12%), respectively. Twelve subjects (12%) carried two defect alleles (*2/*2, *2/*3 or *3/*3), 43 subjects (42%) were heterozygous for a mutated (*2 or *3) and a wild type (*1) allele, and the remaining 48 subjects (47%) were homozygous for the wild type allele. The distributions of the metabolic ratio between these three genotype groups were significantly different (Kruskal-Wallis test: p < 0.0001). The genotypes of 19 additional Korean PMs has been identified in a previous mephenytoin study. From a total of 32 PMs, 31 were genotypically PMs by analysis of the CYP2C19*2 and *3 alleles and only one PM subject was found to be heterozygous for the *1 and *2 alleles. At present it cannot be judged whether this subject has a defective allele with a so-far unidentified mutation or a true wild type allele. We thus confirm a high incidence (12.6%) of PMs of omeprazole in Koreans and of the 32 Korean PMs 97% could be identified by the genotype analysis.

Acceleration of the alcohol oxidation rate in rats with aloin, a quinone derivative of Aloe.

Aloe contains abundant aloin, a C-glycoside derivative of anthraquinone. Based on recent reports indicating that the water extract of Aloe enhances the ethanol oxidation rate and also that quinones, in general, have a functional role in elevating the alcohol oxidation rate in vivo, we have attempted to identify the quinone derivative contained in Aloe that could increase the alcohol oxidation rate. Upon oral administration of aloin (300 mg/kg) given 12 hr prior to the administration of alcohol (3.0 g/kg), the blood alcohol area under the curve (AUC) was found to be decreased significantly (by 40%). This was supported by increases in the rates of blood alcohol elimination and the disappearance of alcohol from the body by 45 and 50%, respectively. Analysis of hepatic triglyceride (TG) levels revealed that both the ethanol and the aloin treatment alone significantly increased the TG levels in a comparable manner; however, the level obtained by the combined treatment of aloin and ethanol was not statistically different from that produced by either treatment alone. The levels of serum L-aspartate:2-oxoglutarate aminotransferase (AST) and L-alanine:2-oxoglutarate aminotransferase (ALT) activities were not increased by acute alcohol intoxication, aloin alone, or by the combined treatment of alcohol and aloin. Pretreatments with aloe-emodin, the anthraquinone aglycone of aloin, resulted in a significantly decreased blood alcohol AUC and an increase in the rate of ethanol disappearance. These results suggested that when the aloin localized primarily in the skin of Aloe is ingested, aloe-emodin (the quinone aglycone) may be released, and the released quinone may produce acceleration of the ethanol metabolism rate in vivo.