PubMed Health⌕ Search

Biomedical subjects

O Pelkonen

Publications and source records attributed to O Pelkonen.

At least 109 records · Page 6Linked to original sources
PubMed1994

Interindividual variability of coumarin 7-hydroxylation in a Turkish population.

One hundred healthy Turkish volunteers (70 male, 30 female) aged from 19 to 56 years were given 5 mg coumarin p.o. after an overnight fast. Urine samples were collected before and 2, 4 and 8 h after drug administration. The extent and rate of formation of 7-OH-coumarin (7OHC) was determined by the urinary excretion of the metabolite as measured with the fluorometric method. On average, 80% of 7OHC formed was excreted in 2 h. The total amount of 7OHC formed was 59.8% (21.5%) (mean and SD, n = 100, range 17-100%) of the given dose. The percentage of 7OHC excreted during the first 2 h compared with the 7OHC excretion at 8 h was a constant and stable individual characteristic for the rate of the formation of 7OHC ('2 h coumarin test'). Although four individuals had relatively slow coumarin test values (34-40%), no clear-cut polymorphism in the rate of 7OHC formation was found. However, 7OHC formation was lower in males and in cigarette smokers.

Adult↗
PubMed1994

Effects of heme arginate on cytochrome P450-mediated metabolism of drugs in patients with variegate porphyria and in healthy men.

We investigated the effects of heme on metabolism of coumarin, debrisoquin, caffeine, and lidocaine in seven female patients with variegate porphyria and in 10 healthy men. During baseline conditions metabolism of the drugs was identical in the two groups. Compared with the results without heme, a single infusion of heme arginate (3 mg/kg heme) significantly decreased the debrisoquin/4-hydroxy-debrisoquin metabolic ratio in subjects with porphyria (p = 0.016) and in the control subjects (p = 0.016) and increased formation of monoethylglycinexylidide from lidocaine (p = 0.016 and p = 0.004, respectively). Metabolism of coumarin and caffeine was not affected by heme. Our results show that, in patients with porphyria and in healthy subjects, exogenous heme is able to accelerate the reactions mediated by the cytochrome isozymes CYP2D6 (debrisoquin) and CYP3A4 (lidocaine) but not reactions mediated by CYP1A2 (caffeine) and CYP2A6 (coumarin). This suggests that influence of heme on drug metabolism is P450 isozyme-specific.

Adult↗
PubMed1994

The expression and environmental regulation of P450 enzymes in human placenta.

The human placenta oxidizes several xenobiotics, although the spectrum of substrates and metabolic activities when compared with the liver appears somewhat restricted. Maternal cigarette smoking or PCB exposure increases the expression of CYP1A1. This induced activity is able to catalyze the activation of benzo(a)pyrene (B(a)P) into DNA-bound products, both in vitro and in vivo. Studies on adult human liver bolster the concept that CYP1A1 and -1A2 are differentially expressed in hepatic and extrahepatic tissues. Studies with cDNA probe or enzyme specific antibodies and substrates for CYP2A, -2B, -2C, -2D, and -2E gene products have yielded negative results. There are only minimal activities that can be found in substantial quantities in placentas without maternal smoking; one example is 7-ethoxycoumarin O-deethylase (ECOD). Aromatase and cholesterol side-chain cleaving P450 mRNAs, proteins, and activities are measurable in human placentas and do not seem to be affected by maternal cigarette smoking.

Cytochrome P-450 Enzyme System↗
PubMed1993

Expression of human placental cytochrome P450 aromatase (CYP19) cDNA in insect cells using a luciferase based baculovirus vector.

The cDNA encoding human placental cytochrome P450 aromatase (CYP19) was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression vector system. The recombinant protein product was characterized by Northern and Western blot analyses as well as by direct measurement of aromatase activity. The expressed enzyme proved to be both catalytically active in the presence of P450 reductase and immunologically reactive with polyclonal antibodies raised against human placental aromatase. The activity of aromatase increased 10% after the addition of 0.1 microgram/ml hemin chloride to the culture medium. However, the level of aromatase protein decreased considerably when the concentration of hemin chloride reached 10 micrograms/ml indicating that hemin chloride has toxic effects on the lepidopteran insect cell line. In conclusion, the baculovirus system is suitable for high level expression of functional human placental CYP19.

Animals↗
PubMed1993

Retrovirus-mediated stable expression of human CYP2A6 in mammalian cells.

To study the pharmacological and toxicological significance of the human cytochrome P450 isoform CYP2A6, we expressed it in mammalian cells by retrovirus-mediated gene transfer. The LXSN vector and PA317 packaging cells were used to create amphotropic recombinant retroviruses containing CYP2A6 cDNA. NIH 3T3 and HeLa cells were infected with these retroviruses and cell clones expressing CYP2A6 were selected. The integration of the CYP2A6 construct was verified by PCR analysis and northern blot analysis showed that a 5 kb mRNA containing the CYP2A6 was present in the cells. The integrated cDNA directed the expression of catalytically active CYP2A6 enzyme which has remained stable over numerous cell passages. No oxidation of several other P450 substrates was detected. The promutagen aflatoxin B1 was metabolized to intermediates binding to the host cell genomic DNA by the 3T3 2A6 cells. These cell lines are thus well suited for the study of the catalytic profile and the biological consequences of promutagen activation by the human CYP2A6 isoform.

3T3 Cells↗
PubMed1993

Differential inhibition of coumarin 7-hydroxylase activity in mouse and human liver microsomes.

Coumarin is 7-hydroxylated by the P450 isoform Cyp2a-5 in mice and CYP2A6 in humans. Various drugs, endogenous substances, plant substances and carcinogens, altogether about 90 chemicals, were evaluated as possible inhibitors of coumarin 7-hydroxylase (COH) activity in mouse microsomes. The effects of selected compounds on COH activity in human liver microsomes were also tested. The furanocoumarin derivatives methoxsalen (8-methoxypsoralen) and psoralen proved to be the most potent inhibitors of mouse COH activity (IC50 values 1.0 and 3.1 microM, respectively). The furanocoumarins bergapten (5-methoxypsoralen), isopimpinellin (5,8-dimethoxypsoralen), imperatorin and sphondin also effectively inhibited mouse COH activity (IC50 values 19-40 microM). Methoxsalen, isopimpinellin and metyrapone were also inhibitors in mice in vivo. Methoxsalen was a potent inhibitor of COH activity also in human liver microsomes, (IC50 value 5.4 microM), whereas bergapten, isopimpinellin and imperatorin had no effect. The imidazole antimycotic miconazole was a potent but non-specific inhibitor of COH activity. Several known substrates and inhibitors of members in the CYP1A, CYP2B, CYP2C, CYP2D and CYP3A subfamilies were poor inhibitors of COH activity. These results suggest that (i) the coumarin-type compounds in particular interact with the active sites of Cyp2a-5 and CYP2A6, and (ii) the active sites of Cyp2a-5 and CYP2A6 are structurally different, since a number of compounds inhibited mouse, but not human COH activity.

Animals↗
PubMed1993

Cytochrome P450 isoforms in human fetal tissues related to phenobarbital-inducible forms in the mouse.

Four polyclonal antibodies raised against purified mouse liver cytochrome P450s representing Cyp1a, Cyp2a, Cyp2b and Cyp2c subfamilies were used to detect their related forms in human adult and fetal tissues. In immunoblot analysis, anti-Cyp2c antibody detected two to three proteins in adult livers and one to three proteins in 70% of the 18 fetal livers studied. Anti-Cyp2a-5 antibody recognized a 50-kDa protein in 50% of the fetal adrenals. Anti-Cyp1a-2 antibody reacted with a single protein (55 kDa) in adult liver. The anti-Cyp2b-10 antibody did not detect proteins in any of the tissues. No proteins were detected in fetal kidneys. There was no coumarin 7-hydroxylase activity (COH) in fetal liver or adrenals. The 7-ethoxycoumarin O-deethylase (ECOD) activities were slightly higher in fetal adrenals (mean 6.1 pmol/mg protein/min) vs livers. The fetal adrenal ECOD activity was not inhibited by the anti-Cyp2a-5 antibody. Aryl hydrocarbon hydroxylase (AHH) activities in fetal livers were about 5% of those in adult livers. AHH activity in fetal liver was not inhibited by the anti-Cyp2c antibody. Testosterone 6 beta-hydroxylase activity was much lower in fetal liver than in adult liver (about 20 and 1700 pmol/mg protein/min, respectively). No immunoinhibition occurred in fetal adrenal progesterone hydroxylation, hepatic benzphetamine N-demethylation and hepatic ethylmorphine N-demethylation. These data suggest that members of the P450 subfamilies 1A, 2A and 2B are expressed at a very low level in fetal liver, and that fetal liver may contain members of the 2C subfamily.

Adrenal Glands↗
PubMed1993

Response of mouse liver coumarin 7-hydroxylase activity to hepatotoxins: dependence on strain and agent and comparison to other monooxygenases.

Acute effects of a single intraperitoneal dose of allyl alcohol (AA, 64 mg/kg), dimethylnitrosamine (DMNA, 30 mg/kg), hexachlorobutadiene (HCBD, 50 mg/kg), carbon tetrachloride (CCl4, 24 mg/kg), cocaine (60 mg/kg) and pyrazole (300 mg/kg) on the hepatic histology and monooxygenases in DBA/2 and C57Bl/6 strains of mice were investigated. All substances caused histologically verified injury to the liver, which varied in appearance and severity depending on the compound and the mouse strain. Responses of P450-catalyzed reactions were highly dependent on the toxin and varied between different monooxygenase (MO) reactions and two mouse strains. In DBA/2 strain, coumarin 7-hydroxylase (COH) activity was increased from 3- to 5-fold by pyrazole, cocaine, HCBD and CCl4. With respect to P450 content and other MO activities, no changes or even decreases were generally observed. Some exceptions to this rule were found: HCBD significantly increased T15 alpha OH, PROD and EROD activities in C57Bl/6 mice, whereas cocaine caused a significant stimulation of T15 alpha OH and PROD in DBA/2 mice, It is concluded that i) different hepatoxins cause different types of liver injury and responses of the monooxygenase complex ("hepatotoxinspecific finger prints"), ii) although DBA/2 and C57Bl/6 mice responded rather similarly to hepatotoxins, also with respect to P450 content and most MO activities, they displayed a profound difference in the behaviour of COH activity, and iii) within the P450 superfamily, the regulation of COH activity seems to be rather unique, also when compared to its structurally close enzyme, testosterone 15 alpha-hydroxylase.

Animals↗
PubMed1993

HPLC method for rapid determination of acetylator phenotype by measuring urinary caffeine metabolites.

A validated reversed-phase high-performance liquid chromatographic (RP-HPLC) method is developed for the selective and rapid determination of two major metabolites of caffeine, namely 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine (MX) from human urine. HPLC separation is achieved by means of a Supersphere-60 RP-Select B (4 microns) analytical column using a non-linear gradient elution programme of 70-95% solvent B (2.5% acetic acid-methanol, 60:40, v/v) in solvent A (water-acetonitrile, 80:20, v/v). A selective UV detection method is used for determination of AFMU, MX and internal standard with readings at 284, 268 and 248 nm, respectively. Urine samples are prepared for measurement by a simple chloroform-diethyl ether (80:20, v/v) extraction. The assay is validated with respect to linearity, sensitivity, accuracy, precision and system suitability. All validation parameters are found to be within the required limits. The limit of detection of AFMU and MX is found to be 50 ng/200 microliters urine. Calibration curves show good linearity between 0.1 and 5 micrograms/200 microliters urine concentration range for both metabolites. The assay is sufficiently sensitive and rapid (4.5 min chromatographic run) to be applied for routine monitoring of change in AFMU/MX molar ratio, indicating acetylation phenotype and change of caffeine metabolism in clinical cocktail studies.

Caffeine↗
PubMed1993

The role of cytochrome P450 3A (CYP3A) isoform(s) in oxidative metabolism of testosterone and benzphetamine in human adult and fetal liver.

Testosterone metabolism was studied in human adult and fetal liver microsomes. In fetal livers 6 beta-hydroxylase (6 beta OH) activity (1-2% of adult activity) and 2 alpha-hydroxylase (2 alpha OH) activity (about 40% of adult activity) were present. Also some fetal livers produced two unknown metabolites. Androstenedione was formed in all fetal livers studied (10-20% of adult activity). Testosterone hydroxylations at 6 beta-, 2 beta-, 15 alpha- and 15 beta-positions were associated with CYP3A isoform(s) in adult liver, because they were strongly inhibited by midazolam, a known substrate for CYP3A4 and by anti-CYP3A4 antibody. Fetal liver activities were consistently inhibited less than the activities in adult livers. The formation of androstenedione was not affected by these inhibitors in fetal or adult liver microsomes. Benzphetamine N-demethylase activity in the fetal livers was about 40% of adult activity. Anti-CYP3A4 antibody had no effect on that activity in fetal or in adult liver microsomes, whereas a monoclonal antibody 1-68-11 (generated against rat CYP2C11) slightly inhibited benzphetamine N-demethylase activity in adult liver. This study indicates that human fetal and adult liver are dissimilar in their testosterone metabolism pattern. The formation of androstenedione from testosterone in fetal liver may have a physiological role. Testosterone hydroxylases are less inhibited by anti-CYP3A4 antibody, midazolam and progesterone in fetal than in adult liver.

Adult↗
PubMed1993

PCR-based CYP2D6 genotyping for Finnish lung cancer patients.

Polymorphism of the gene encoding for debrisoquine hydroxylase, i.e. CYP2D6, was determined genotypically for 122 healthy controls and 106 lung cancer patients using Xba I restriction fragment length polymorphism (RFLP) analysis, together with a combination of two recently published polymerase chain reaction (PCR) based approaches. Three different mutated alleles of the CYP2D6 gene were detected; CYP2D6B comprised 11.1% and 10.4% of the total alleles in the controls and in the lung cancer patients, CYP2D6A had frequencies of 5.7% and 2.8%, and CYP2D6D had frequencies of 3.3% and 2.4%, respectively. Only 17 of the 24 44 kb Xba I alleles (71%) were confirmed as defective alleles carrying the mutation in CYP2D6B loci, whereas all four 15 + 9 kb Xba I alleles contained the CYP2D6B mutation. Out of the 122 healthy controls, seven subjects (5.7%) were detected as poor metabolizers (PMs) of debrisoquine by the presence of two defective alleles, whereas only one PM genotype was found in the lung cancer patient group (0.9%). The reliability of this analysis was confirmed in a subgroup of the control subjects phenotyped by debrisoquine, where a perfect correlation between CYP2D6 phenotype and genotype was obtained. We observed no significant difference in the allelic frequencies between lung cancer patients with a history of heavy smoking and those who smoked less. However, statistical analysis showed a significant difference (p = 0.05) in distribution of the PM-associated genotypes between lung cancer patients (1/106) and healthy controls (7/122). This data thus supports the hypothesis that there is an increased risk of lung cancer for individuals who are extensive metabolizers of debrisoquine.

Aged↗
PubMed1993

Cytochrome P4502A-mediated coumarin 7-hydroxylation and testosterone hydroxylation in mouse and rat lung.

Pulmonary coumarin 7-hydroxylase, testosterone hydroxylase and other P450-mediated activities were compared in the mouse and rat. Coumarin 7-hydroxylase activity was 20 pmol/mg/min. in mouse and 4 pmol/mg/min. in rat lung microsomes. Liver values were 180 (mouse) and 1 (rat) pmol/mg/min. Km values of rat and mouse lung coumarin 7-hydroxylase were about 1 microM whereas the rat liver Km value was > 100 microM. Phenobarbital and 3-methylcholanthrene did not affect rat lung (or liver) coumarin 7-hydroxylase activity. Anti-Cyp2a-5 antibody effectively inhibited mouse and rat lung coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylations but failed to block these activities in the rat liver. In immunoblot analysis anti-Cyp2a-5 antibody recognized the 50-kDa Cyp2a-4/5 protein in mouse lung microsomes. A P450 protein co-migrating with Cyp2a-5 was also detected in rat lung microsomes. Cyp2a-5 cDNA probe hybridized with a 1.8-kb mRNA species in rat lung RNA fraction. The hybridization signal was not increased by 3-methylcholanthrene or phenobarbital. These data suggest that the mouse lung expresses Cyp2a-5 which differs from the liver enzyme only in its regulation and that the rat lung contains a P450 isoform(s) belonging to the 2A subfamily which may be orthologous with the mouse Cyp2a-4/5 catalyzing coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylase activities. The recently reported rat lung CYP2A3 (Kimura et al.) gene product is a candidate for the observed coumarin 7-hydroxylase activity in the rat lung.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1993

Involvement of P450 1A1 in benzo(a)pyrene but not in benzo(a)pyrene-7,8-dihydrodiol activation by 3-methylcholanthrene-induced mouse liver microsomes.

Synchronous fluorescence spectrophotometry for benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adducts was used to study the activation pathway of benzo(a)pyrene in C57BL/6 mice. Benzo(a)pyrene but not benzo(a)pyrene-7,8-diol activation by 3-methylcholanthrene-induced mouse liver microsomes was inhibited by a monoclonal antibody (Mab 1-7-1) against CYP1A1/2 suggesting that 1A1 probably takes part in the first P450 reaction. However, aryl hydrocarbon hydroxylase activity, a classical measure of benzo(a)pyrene metabolism, was not inhibited by the same concentration of Mab 1-7-1. None of the other antibodies used, detecting 2A, 2B, 2C or 2E subfamilies, inhibited the adduct formation. Troleandomycin and gestodene, chemical inhibitors of human 3A4, inhibited benzo(a)pyrene-7,8-diol activation by 3-methylcholanthrene-induced microsomes to some extent only in high concentrations. Although liver microsomes from 3-methylcholanthrene-induced mice catalyzed the formation of BPDE-DNA in vitro clearly more than uninduced microsomes, 3-methylcholanthrene pretreatment in vivo decreased the adduct formation in benzo(a)pyrene-treated mice. These results emphasize the significance of detoxicating and DNA-repairing pathways in vivo. Finally, synchronous fluorescence spectrophotometry for BPDE-DNA measures the end-point of the three-step activation pathway while aryl hydrocarbon hydroxylase measures a one-step hydroxylation. Thus, these methods should be used rather as corroborative than mutually exclusive assays.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1992

Cerium-induced strain-dependent increase in Cyp2a-4/5 (cytochrome P4502a-4/5) expression in the liver and kidneys of inbred mice.

The murine Cyp2a-4 and Cyp2a-5 genes encode P450 isoforms catalysing testosterone 15 alpha-hydroxylase and coumarin 7-hydroxylase (COH) activities, respectively. Two days after the administration of a hepatotoxic dose of cerium chloride (2 mg/kg i.v.), COH activity was increased 3.2-fold in the liver of DBA/2 mice. Three and 4 days after the cerium treatment, coinciding with the occurrence of overt liver damage, there was a dramatic decrease in COH activity. The activities of testosterone 15 alpha-hydroxylase and the Cyp1a-1-mediated 7-ethoxyresorufin O-deethylase (EROD) were decreased in response to cerium. Much less pronounced changes in the enzyme activities occurred in the C57BL/6 mouse liver. Northern blot analysis showed a 21-fold increase in the hepatic Cyp2a-4/5 mRNA in the DBA/2 mice at day 2, whereas no increase occurred in the C57BL/6 mice. Also in the kidneys the increase in COH activity and in Cyp2a-4/5 mRNA was marked only in the DBA/2 mice. A polymerase chain reaction-mediated analysis method utilizing a unique PstI restriction site in the Cyp2a-5 cDNA was used to differentiate between the highly homologous Cyp2a-4 and Cyp2a-5 mRNAs. Cerium was found to increase the amount of hepatic and renal Cyp2a-4 and Cyp2a-5 mRNA only in the DBA/2 mice. These data indicate that the Cyp2a-4/5 complex is regulated in a different way in DBA/2 and C57BL/6 mice and that some association exists between the development of liver damage and COH induction.

Animals↗
PubMed1992

Effect of pyrazole, cobalt and phenobarbital on mouse liver cytochrome P-450 2a-4/5 (Cyp2a-4/5) expression.

Pyrazole, cobalt and phenobarbital increase the activity of coumarin 7-hydroxylase (COH) in mouse liver. To study the mechanism of this increase, we measured the expression of the cytochrome P-450 2a-4/5 (Cyp2a-4/5) complex, which mediates testosterone 15 alpha-hydroxylase and COH activities, as a function of dose and time after the treatment of C57BL/6 (B6) and DBA/2 (D2) male mice with the inducers. COH activity and Cyp2a-4/5 steady-state mRNA levels were increased in both strains in response to the inducers. No marked effect occurred with testosterone 15 alpha-hydroxylase or activities associated with Cyp1a-1 or Cyp2e-1. A 2-7-fold increase in response to the inducers was seen in the amount of P-450Coh (cytochrome P-450 isoenzyme catalysing coumarin 7-hydroxylation) protein in Western immunoblots. PCR amplification of a 1 kb region in Cyp2a-4/5-mRNA-derived cDNA, followed by cutting at the diagnostic PstI site, showed that most of the steady-state mRNA consisted of Cyp2a-5, which is also the form most affected by pyrazole. Nuclear run-off analysis revealed no increase in the transcription rate of Cyp2a-4/5 after pyrazole or cobalt treatment, whereas a 2-3-fold increase occurred after phenobarbital pretreatment in B6 mice. Together with previous reports [Aida & Negishi (1991) Biochemistry 30, 8041-8045], the current data suggest that both pyrazole and cobalt increase COH catalytic activity by affecting Cyp2a-5 by post-transcriptional mechanisms in mice.

Animals↗
PubMed1992

Interindividual variability of coumarin 7-hydroxylation in healthy volunteers.

A test designed to estimate the extent and rate of formation of 7-hydroxycoumarin by measuring the urinary excretion of the metabolite in humans after administering 5 mg coumarin was developed. Coumarin was rapidly metabolized after oral administration and more than 95% of the 7OHC formed was excreted in 4 h. The total amount of 7OHC formed was 64 +/- 15% (mean +/- SD, variation 20-100%) of the dose given. The percentage of 7OHC excreted in 2 h, as compared with the 7OHC excretion in 4 h, was found to be a constant and stable individual characteristic for the rate of the formation of 7OHC ('2 h coumarin test'). In 110 volunteers, there was a great interindividual variability in the extent and rate of 7OHC formation. Four individuals had relatively 'slow' coumarin test values (50-60%), but much larger populations would be needed for the demonstration of polymorphism.

Adult↗