[Do patients react strangely to drugs? Knowledge of genetic aberrations increases slowly. Interview by Tord Ajanki].
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Chromosome Aberrations↗
Biomedical subjects
I Johansson
Publications and source records attributed to I Johansson.
In vitro kinetics of two human CYP1A1 variant enzymes suggested to be associated with interindividual differences in cancer susceptibility.
A genetic polymorphism (A4889-->G) in the human CYP1A1 gene which creates an Ile462-->Val amino acid substitution has been suggested to cause altered enzymatic properties of CYP1A1. Since several epidemiological studies have shown an association between the CYP1A1-Val allele and lung cancer, we considered it of importance to evaluate the in vitro kinetic properties of the two CYP1A1 variants after expression of each cDNA in yeast. No differences were found in K(m) or Vmax for CYP1A1 dependent O-dealkylation of ethoxyresorufin and 3-hydroxylation of benzo(a)pyrene between the two variants. The data indicate that the Ile/Val polymorphism in human CYP1A1 is not functionally important.
Benzo(a)pyrene↗
The involvement of CYP1A2 and CYP3A4 in the metabolism of clozapine.
AIMS: Clozapine (CLZ), an atypical neuroleptic with a high risk of causing agranulocytosis, is metabolized in the liver to desmethylclozapine (DCLZ) and clozapine N-oxide (CLZ-NO). This study investigated the involvement of different CYP isoforms in the formation of these two metabolites. METHODS: Human liver microsomal incubations, chemical inhibitors, specific antibodies, and different cytochrome P450 expression systems were used. RESULTS: Km and Vmax values determined in human liver microsomes were lower for the demethylation (61 +/- 21 microM, 159 +/- 42 pmol min(-1) mg protein(-1) mean +/- s.d.; n = 4), than for the N-oxidation of CLZ (308 +/- 1.5 microM, 456 +/- 167 pmol min(-1) mg protein(-1); n = 3). Formation of DCLZ was inhibited by fluvoxamine (53 +/- 28% at 10 microM), triacetyloleandomycin (33 +/- 15% at 10 microM), and ketoconazole (51 +/- 28% at 2 microM) and by antibodies against CYP1A2 and CYP3A4. CLZ-NO formation was inhibited by triacetyloleandomycin (34 +/- 16% at 10 microM) and ketoconazole (51 +/- 13% at 2 microM), and by antibodies against CYP3A4. There was a significant correlation between CYP3A content and DCLZ formation in microsomes from 15 human livers (r=0.67; P=0.04). A high but not significant correlation coefficient was found for CYP3A content and CLZ-NO formation (r=0.59; P=0.09). Using expression systems it was shown that CYP1A2 and CYP3A4 formed DCLZ and CLZ-NO. Km and Vmax values were lower in the CYP1A2 expression system compared to CYP3A4 for both metabolic reactions. CONCLUSIONS: It is concluded that CYP1A2 and CYP3A4 are involved in the demethylation of CLZ and CYP3A4 in the N-oxidation of CLZ. Close monitoring of CLZ plasma levels is recommended in patients treated at the same time with other drugs affecting these two enzymes.
Antibodies↗
Depressive symptoms in individuals with idiopathic subjective dry mouth.
It has been known for many centuries that there is a relationship between saliva flow rate and emotional status. The significance of psychological processes in the subjective sensation of a dry mouth has been discussed earlier, and this study deals with the presence of depressive symptoms in individuals with idiopathic subjective sensation of a dry mouth. Depressive symptoms in 94 healthy subjects with normal flow rates for unstimulated and stimulated whole saliva but with a subjective sensation of a dry mouth were assessed by the Beck Depression Inventory (BDI) and compared with healthy age- and gender-matched controls. The subjects with a subjective dry mouth condition were significantly more depressive and also had a significantly higher frequency of depressive symptoms. Depression was found in 21.3% of the individuals with a subjective dry mouth sensation and in 3.2% of the controls. The results of this study indicate that, in some cases, subjective dry mouth may be of psychological origin.
Adult↗
Cardiovascular disease risk factors and dental caries in adolescents: effect of a preventive program in Northern Sweden (the Norsjö project).
Since 1985 a 10 year prevention programme aiming to reduce cardiovascular diseases (CVD) has been running in the county of Västerbotten in Northern Sweden. The project started in Norsjö. The present investigation is a study on dietary intake, medical CVD risk factors and dental caries in five cross-sectional groups of 15-year-olds during 5 years (1987-1991) of the "Norsjö project". Most of the measured medical and dietary variables followed a similar trend, i.e. a positive trend during the first 3 years (1987-1989) and in the last 2 years (1990-1991) the averages returned towards baseline values. Dental caries prevalence followed a similar trend. Parental educational level did not have a major influence on diet or medical CVD risk factors, but higher caries scores were noted in adolescents with parents with "low" education compared with adolescents where the parents had higher educational levels. The results from the study also point to the fact that dental caries prevalence together with body mass index may indicate adolescents with CVD risk factors at unfavourable levels. Dietary counselling by dental personnel to adolescents with high caries and moderate obesity can be of advantage in reducing caries risk, as well as risk for development of CVD at higher ages.
Adolescent↗
A combination of mutations in the CYP2D6*17 (CYP2D6Z) allele causes alterations in enzyme function.
In many black African populations, the capacity for CYP2D6-dependent drug metabolism is generally reduced. A specific variant of the CYP2D6 gene (CYP2D6*17) that carries three functional mutations (T107I, R296C, and S486T) has been found to be present in Zimbabwean subjects with impaired CYP2D6-dependent hydroxylase activity. To evaluate whether the CYP2D6*17 allele was the major cause behind the decreased rate of drug metabolism and to examine the role of the different mutations, CYP2D6 cDNAs containing all eight combinations of the mutations were created. Expression of the cDNAs in COS-1 cells revealed that the CYP2D6 17 enzyme displayed only 20% of the wild-type (CYP2D6 1) activity, whereas the T107I substitution on its own had no significant effect on enzyme function. Expression in yeast showed that the three possible single amino-acid mutant CYP2D6 variants all had properties similar to CYP2D6 1 when the kinetics of bufuralol hydroxylation was examined. However, enzymes containing both the T107I and R296C mutations exhibited a more than 5-fold higher K(m) for bufuralol than the wild-type enzyme, whereas the S486T mutation was of little importance. In contrast, when codeine was used as a substrate, the T107I substitution alone was sufficient to cause a significant increase in the apparent K(m), indicating a differential effect for this substitution depending on the CYP2D6 substrate. In conclusion, the CYP2D6*17 allele represents the first human cytochrome P450 polymorphic variant in which a combination of substitutions is required to alter the enzyme's catalytic properties and is the first case in which a decreased CYP2D6 activity, as monitored in vivo, has been documented to be caused by an enzyme with altered affinity for CYP2D6 substrates.
Adrenergic beta-Antagonists↗
Salivary cholesterol of healthy adults in relation to serum cholesterol concentration and oral health.
Salivary lipids are mostly glandular in origin, but some are believed to diffuse directly from serum. This diffusion and the role of salivary lipids in oral health have scarcely been studied. Therefore, the serum and saliva cholesterol concentrations and oral health were analyzed in a group of healthy adults (n = 139; 64 men and 75 women; 34.2 +/- 5.2 yrs). Paraffin-stimulated whole saliva was collected, centrifuged (10,000 x g; 30 min, 4 degrees C), and lyophilized, and the cholesterol and other neutral lipids were extracted, separated by thin-layer chromatography, and quantified. The mean +/- SD (range) of saliva cholesterol concentration was 1.20 +/- 0.75 (0.02-5.46) mumol/L, and the saliva cholesterol level of men (1.36 +/- 0.85 mumol/L) was significantly higher than that of women (1.06 +/- 0.64 mumol/L; p < 0.05). Weak positive correlations between saliva and serum cholesterol concentrations and saliva cholesterol and serum non-high-density lipoprotein cholesterol concentrations were found (r = 0.22, p < 0.05; r = 0.28, p < 0.005, respectively). The saliva cholesterol assay detected subjects with high (> or = 6.5 mmol/L) serum cholesterol values, with sensitivity and specificity values of 100% and 29%, respectively. A positive correlation between the body mass index and the level of saliva cholesterol concentration was also found (r = 0.31 p < 0.01). Oral health, microbial counts, or saliva flow rate revealed no differences in subjects with low and high salivary cholesterol level. We conclude that, in healthy adults, saliva cholesterol concentration reflects serum concentration to some extent and can be used to select individuals with high serum cholesterol levels.
Adult↗
Rat salivary gland function after fractionated irradiation.
The aim of this study was to investigate longitudinal effects of fractionated irradiation, with various total doses, on salivary gland function in the rat. Female Sprague-Dawley rats were irradiated with 4, 5, 6, 7 or 8 Gy per day on five consecutive days. Irradiation was given to the head and neck region. Whole saliva was collected before and 2, 15 and 26 weeks after irradiation. In general the effects of irradiation on salivary gland function were found to be related to dose and time after exposure. Secretion rates were significantly decreased two weeks after irradiation with doses of 30 Gy or higher, after 15 weeks with 25 Gy or higher, and after 26 weeks with 20 Gy or higher. Response patterns to irradiation differed between the salivary constituents. Thus, the conclusions from this study are that early and late effects display different patterns and that the model used to study variations in salivary gland function after fractionated irradiation must be adjusted to the question addressed.
Animals↗
Selective centrilobular expression of the aryl hydrocarbon receptor in rat liver.
The aryl hydrocarbon receptor (AHR) is a transcriptional activator of genes encoding a group of drug-metabolizing enzymes, including cytochrome P450 1A1 (CYP1A1), glutathione S-transferase, tumor-associated aldehyde dehydrogenase and quinone reductase. Both the constitutive and inducible expression of these genes in the liver is zonated, i.e., dominant in hepatocytes of the centrilobular region, a poorly understood position-dependent phenomenon. By comparing cell lysates obtained from opposite acinar regions we observed that immunoreactive AHR protein was almost exclusively confined to centrilobular cells. The AHR mRNA, as analyzed from cell lysates by reverse transcriptase polymerase chain reaction, exhibited a similar, although somewhat less pronounced zonation. By contrast, only slight zonation of the AHR nuclear translocator mRNA was observed. Treatment of rats with omeprazole, an atypical nonligand activator of the AHR, caused a zone-specific induction of CYP1A1 in the centrilobular region similar to that seen after pretreatment with the AHR ligand 3-methylcholanthrene. Our results suggest that the zone-restricted expression of AHR protein will allow the constitutive and inducible expression of AHR-regulated genes in the centrilobular region, but will limit their expression in the periportal region.
Animals↗
Genetic polymorphism of human CYP2E1: characterization of two variant alleles.
Ethanol-inducible CYP2E1 is an enzyme of major toxicological interest because it metabolizes several precarcinogens, drugs, and solvents to reactive metabolites. CYP2E1 has also been implicated in alcohol liver disease because of its contribution to oxidative stress. Previously, polymorphic alleles with mutations in introns and in the 5'-flanking regulatory region have been described, and their presence has been related to the incidence of alcohol liver disease and lung cancer. In the present investigation, we investigated whether any functional mutations are linked to the above-mentioned rare alleles and also screened for mutations in the open reading frame using single-stranded conformation polymorphism and genomic DNA from almost 200 individuals belonging to either a Chinese, an Italian, or a Swedish population. Two new CYP2E1 gene variants were found with functional mutations: one (CYP2E1*2) in which a G1168A point mutation in exon 2 caused an R76H amino acid substitution, and the other (CYP2E1*3) in which a G10059A base substitution in exon 8 yielded a V3891 amino acid exchange. The corresponding CYP2E1 cDNAs were constructed, subcloned into the pCMV4 expression vector, and expressed in COS-1 cells. The cellular levels of CYP2E1 mRNA, CYP2E1 protein, and rate of chlorzoxazone hydroxylation were monitored. The CYP2E1*3 cDNA variant was indistinguishable from the wild-type cDNA on all variables investigated, whereas CYP2E1*2 cDNA, although yielding similar amounts of mRNA, only caused 37% of the protein expression and 36% of the catalytic activity compared with the wild-type cDNA. Complete screening by single-stranded conformation polymorphism of the three populations studied revealed that these variant alleles were rare. We conclude that the human CYP2E1 gene is functionally surprisingly well conserved compared with other cytochrome P450 enzymes active in drug metabolism, which suggests an important endogenous function in humans.
Alleles↗
Alkyl Glucosides on Hydrophobic Surfaces Studied by Surface Force and Wetting Measurements
The wetting properties of hydrophobized surfaces in the presence of alkyl glucosides have been studied using a Wilhelmy balance technique, and the interparticle forces acting between such surfaces have been investigated using the interferometric surface force apparatus. The adsorption at concentrations well below the cmc is rather limited both for the anomerically pure octyl beta-glucoside and for the technical product based on the branched octyl glucoside, 2-ethylhexyl glucoside. However, the adsorption results in a decreasing surface hydrophobicity, and consequently the force needed to separate two such surfaces from each other is reduced. At the cmc for octyl beta-glucoside, a surfactant monolayer is adsorbed on each surface. The interaction between the glucose units gives rise to a short-range repulsive force. Beyond the range of this repulsive force a weak attraction is experienced. Addition of a small amount of octyl alpha-glucoside does not change the short-range interaction significantly. The technical 2-ethylhexyl glucoside surfactant behaves similarly to the anomerically pure surfactant at low concentrations. The solution with the technical glucoside phase-separates at high surfactant concentration. It is observed that under such conditions a thick layer is deposited on each surface.
Journal Article↗
Anti-adhesion and diagnostic strategies for oro-intestinal bacterial pathogens.
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Animals↗
Phenotype and genotype analysis of debrisoquine hydroxylase (CYP2D6) in a black Zimbabwean population. Reduced enzyme activity and evaluation of metabolic correlation of CYP2D6 probe drugs.
OBJECTIVE: Debrisoquine hydroxylase (CYP2D6) is responsble for the oxidative metabolism of many clinically used drugs. Since this enzyme has been poorly studied in the southern part of Africa, we examined the CYP2D6 phenotypes and genotypes in 103 unrelated black Zimbabweans. METHODS: Phenotyping for CYP2D6 activity was done using debrisoquine and metoprolol as probe drugs by measuring the urinary metabolic ratio (MR) of parent drug to metabolite concentration ratios. Genotyping was done using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP) and sequencing analyses with respect to CYP2D6 variants of interest. RESULTS AND CONCLUSION: Phenotyping with debrisoquine revealed two poor metabolisers (PMs), whereas 5 subjects out of 94 were PMs using metoprolol as probe drug. Genotypes predictive of the poor metaboliser status were observed for the two subjects who were PMs with both probe drugs, whereas no mutations could explain the PM phenotype for metoprolol among the three remaining subjects, a fact possibly explained by lack of compliance in metoprolol intake. There was a moderate correlation of 0.67 between the debrisoquine and metoprolol metabolic ratios in the 89 subjects who were extensive metabolisers for both probe drugs. The median values for the metabolic ratios for debrisoquine and metoprolol as probe drugs were 1.00 and 1.35, respectively, which are higher than those observed in Caucasian populations. This is indicative of a decreased capacity for metabolism of CYP2D6 substrates by Zimbabweans compared to Caucasians. Evaluation of the DNA samples for the known allelic variants CYP2D6A, CYP2D6B, CYP2D6C, CYP2D6D or CYP2D6Ch1 yielded no explanation for these results.
Cytochrome P-450 CYP2D6↗
Hepatotoxicity induced by iron overload and alcohol. Studies on the role of chelatable iron, cytochrome P450 2E1 and lipid peroxidation.
BACKGROUND/AIMS: Clinical experience and studies with experimental animal models indicate a synergistic hepatotoxic effect of dietary iron overload and chronic alcohol ingestion. In order to elucidate the mechanism underlying this synergism, we examined the hepatic levels of ethanol-inducible cytochrome P450 2E1, glutathione and malondialdehyde, and the effect of iron chelation with desferrioxamine, in livers from rats treated with iron and/or ethanol. METHODS: Animals received diets with or without 2.5-3% carbonyl iron for 6-9 weeks, followed by an ethanol-containing diet or a liquid control diet for 5-9 weeks. Desferrioxamine was administered subcutaneously with mini-osmotic pumps. Alanine aminotransferase activity in serum and hepatic contents of glutathione and malondialdehyde were determined. The hepatic level of cytochrome P450 2E1 was determined with Western Blotting using a specific polyclonal antibody. RESULTS: The combination of iron and alcohol led to a marked increase in serum alanine aminotransferase activity as compared with all other treatment groups, and iron chelation with desferrioxamine reversed these increases. Treatment with alcohol alone led to slightly increased aminotransferases compared with controls. The level of cytochrome P450 2E1 was significantly elevated in microsomes isolated from ethanol-treated rats, but neither additional iron supplementation nor desferrioxamine influenced this level significantly. Glutathione contents were increased in the livers of animals treated with iron and/or ethanol. Malondialdehyde values were increased in iron-treated animals, whereas neither ethanol nor desferrioxamine altered malondialdehyde levels significantly. CONCLUSIONS: The toxic effects exerted by the combination of iron overload and chronic ethanol feeding on rat liver are dependent on a pool of chelatable iron. The hepatic level of cytochrome P450 2E1 is markedly induced by ethanol but not further altered by iron overload. Neither increased lipid peroxidation nor depletion of hepatic glutathione levels can explain the synergistic hepatotoxic effects of iron and ethanol in this model.
Alanine Transaminase↗
PCR-based genotyping for duplicated and deleted CYP2D6 genes.
The debrisoquine hydroxylase (CYP2D6), which metabolizes more than 30 different drugs, is highly polymorphic. In subjects having either very low or very high enzyme activity, drug therapy at recommended doses using CYP2D6 substrates may lead to either increased risk of side effects or therapeutic failure. We here describe PCR-based methods for detection of alleles having either duplicated, multiduplicated or deleted active CYP2D6 genes. As a control reaction, the entire coding region of the CYP2D6 gene is amplified. In conjunction with analysis of common mutations using this product as a template, the methods described can be used for genotyping of individuals being either poor, intermediate rapid, normal or ultrarapid metabolizers and provides an efficient tool for individualization of drug therapy.
Cytochrome P-450 CYP2D6↗
Debrisoquine and S-mephenytoin hydroxylation phenotypes and genotypes in a Korean population.
One hundred and fifty-two healthy Korean volunteers were phenotyped with debrisoquine and mephenytoin and genotyped with respect to CYP2D6. The debrisoquine metabolic ratio (MR) varied between 0.09 and 6.3, and all subjects were thus classified as extensive metabolizers of debrisoquine. Polymerase chain reaction (PCR)-based amplification of genomic DNA with primers specific for the C188-->T mutation present in exon 1 of the CYP2D6*10B allele was performed and revealed an allele frequency of 0.51 in this Korean population. Forty-three subjects (28%) were homozygous for CYP2D6*10B, 69 subjects (45%) were heterozygous for this allele, while in 40 subjects (26%) no exon 1 mutation could be found. All subjects except one homozygous for the wild type allele had MRs below 0.75 whereas the MR was higher than 0.99 in all subjects homozygous for the CYP2D6*10B allele. The MRs in the three genotype groups were significantly different (p < 0.0001; Kruskal-Wallis test). Eco RI RFLP analysis of DNA from six subjects with debrisoquine MRs < or = 0.11 revealed that only one (MR 0.09) carried a duplicated CYP2D6*Z-gene (CYP2D6*2X2) as indicated by the Eco RI 12.1 kb haplotype. It is concluded that, as shown earlier for Chinese and Japanese populations, the CYP2D6*10B-allele containing the C188-->T mutation is the major cause of diminished CYP2D6 activity in Koreans. In this Korean population, the MR of debrisoquine was shifted towards higher values (lower CYP2D6 activity) compared with Caucasian populations but the shift appeared to be less pronounced than earlier shown for Chinese. Twenty-four subjects (16%) were poor metabolizers of S-mephenytoin as indicated by the S/R mephenytoin ratio of about 1. Twenty-three of these were genotyped with respect to the defect CYP2C19-alleles CYP2C19*2 and CYP2C19*3. Of the 46 poor metabolizer alleles, 32 (70%) were CYP2C19*2 and the remaining 14 (30%) were CYP2C19*3. Thus, the defect CYP2C19*2 and CYP2C19*3-alleles explained 100% of the 23 Korean poor metabolizers of S-mephenytoin.
Adult↗
The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca2+ and apoplastic water potential.
We show that homologs of the major intrinsic protein (MIP) family are major integral proteins of the spinach leaf plasma membrane and constitute approximately 20% of integral plasma membrane protein. By using oligonucleotide primers based on partial amino acid sequences for polymerase chain reaction and screening of a spinach leaf cDNA library, we obtained two full-length clones of MIP homologs (pm28a and pm28b). One of these clones, pm28a, was sequenced, and it encodes a protein (PM28A) of 281 amino acids with a molecular mass of 29.9 kD. DNA gel blots indicated that PM28A is the product of a single gene, and RNA gel blots showed that pm28a is ubiquitously expressed in the plant. In vivo phosphorylation of the 28-kD polypeptide(s), corresponding to PM28A and PM28B, was dependent on apoplastic water potential, suggesting a role in regulation of cell turgor for these putative aquaporins. In vitro, only one of the homologs, PM28A, was phosphorylated. Phosphorylation of PM28A occurred on Ser-274, seven amino acids from the C terminus of the protein, within a consensus phosphorylation site (Ser-X-Arg) for vertebrate protein kinase C. In vitro phosphorylation of PM28A was due to a plasma membrane-associated protein kinase and was strictly dependent on submicromolar concentrations of Ca2+.
Amino Acid Sequence↗
Saliva composition in children and young adults with Papillon-Lefèvre syndrome.
The aim of the present study was to evaluate the salivary secretion rate and composition in a group of 16 children and young adults (6-27 years) with Papillon-Lefèvre Syndrome (PLS), and to compare the findings with a group (n = 16) of healthy controls. Unstimulated and stimulated whole saliva was collected at least 2 h after meals and the secretion rate determined. The stimulated saliva was assessed for buffer capacity, total protein, peroxidase and hexosamine, while the unstimulated samples were evaluated for total protein, lysozyme, thiocyanate, lactoferrin and salivary IgA. Both the unstimulated (p < 0.01) and stimulated (p < 0.05) saliva secretion rates were significantly lower among the PLS patients compared with the controls. Furthermore salivary buffer capacity was significantly (p < 0.01) lower in the PLS patients. The total protein content in saliva was comparatively high in the study group, while the concentrations of immunoglobulins and non-immunoglobulins were within normal ranges. When calculating the output of the assessed antimicrobial factors, the mean peroxidase level in stimulated whole saliva was found to be significantly (p < 0.01) lower in the PLS patients than in the healthy controls. In conclusion, the present study indicates an impaired water secretion and a somewhat altered saliva gland function in children and young adults with PLS.
Adolescent↗