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M Ingelman-Sundberg

Publications and source records attributed to M Ingelman-Sundberg.

At least 37 records · Page 2Linked to original sources
PubMed2001

Characterization of the CYP2D6*29 allele commonly present in a black Tanzanian population causing reduced catalytic activity.

Debrisoquine metabolism among Tanzanians has been found to be slower than expected from the CYP2D6 genotype. In order to evaluate any genetic explanation, the coding sequence and intron-exon boundaries of the CYP2D6 gene from three Black Tanzanian volunteers with a CYP2D6*1/*1 or CYP2D6*2/*2 genotype and debrisoquine metabolic ratios (MRs) > 1 were fully sequenced to screen for new mutations. Two functional mutations, G1747 to A (causing V136I) and G3271 to A (causing V338M), were identified in the CYP2D6*2/*2 sample. Thirty-six subjects (34%) out of a total 106 subjects were heterozygous and three subjects (3%) were homozygous for the allele, yielding an allele frequency of 20%. The CYP2D6*29 allele, having also the mutations of the CYP2D6*2 allele, was subsequently expressed in yeast and mammalian COS-1 cells. No differences were seen with respect to the affinity (Km) or maximal velocity (Vmax) of the CYP2D6 substrate bufuralol between the wild-type and mutant when expression was carried out in yeast cells. By contrast, the 1'-hydroxybufuralol catalytic activity of the mutant expressed in COS-1 cells was only 26% of the wild-type (P < 0.01; Mann-Whitney U-test) and its debrisoquine hydroxylation activity was 63% of that of CYP2D6.1. The single mutants V136I and V338M had reduced capacity for bufuralol hydroxylation, but the effect was even stronger when both mutations were present together as in CYP2D6.29. Analysis of the distribution of CYP2D6*29 in subjects phenotyped for debrisoquine revealed that this allele significantly causes a reduction in the rate of debrisoquine hydroxylation in vivo. The results indicate the common existence in Tanzanians of a variant CYP2D6 form with different substrate specificity as compared to the wild-type form of the enzyme causing reduced capacity for debrisoquine metabolism.

Alleles↗
PubMed2001

Functional polymorphism in the alcohol dehydrogenase 3 (ADH3) promoter.

The ADH3 gene encodes alcohol dehydrogenase 3 (ADH3)/glutathione-dependent formaldehyde dehydrogenase, the ancestral and most conserved form of alcohol dehydrogenase. ADH3 is expressed in all tissues examined and the enzyme is essential for formaldehyde scavenging. We have screened the promoter region including exon 1 and exons 5, 6 and 7 of the ADH3 gene for allelic variants. Using 80 samples of genomic DNA from Swedes as template, the various parts of the gene were PCR amplified and subsequently analyzed on single strand conformation polymorphism (SSCP) gels. No abnormal migration patterns could be detected by SSCP analysis of exons 5, 6 and 7 while for the promoter region, a large number of the samples displayed differences in SSCP gel migration patterns. Cloning and sequence analysis revealed four possible base pair exchanges in the promoter region. Two transitions were found at position -197 and -196, GG --> AA, one at position -79, G --> A and finally, close to the transcription start site, a fourth transition was found at position +9, C --> T. An allele specific PCR method was developed and allele frequencies were determined in three populations: Chinese, Spanish and Swedish. GG-197,-196 and AA-197,-196 alleles were common in all three populations, G-79 and A-79 were common in Swedes and Spaniards but only A-79 was found among Chinese. T+9 was the most rare allele with an allele frequency of 1.5% in Swedes. Finally, promoter activity assessments and electrophoretic mobility shift assays demonstrated that the C+9 --> T+9 exchange resulted in a significant transcriptional decrease in HeLa cells and a decreased binding of nuclear proteins. These base pair exchanges may have an effect on the expression of the enzyme and thereby influence the capacity of certain individuals to metabolize formaldehyde.

5' Untranslated Regions↗
PubMed2001

Implications of polymorphic cytochrome p450-dependent drug metabolism for drug development.

The main part of human cytochrome P450-dependent drug metabolism is carried out by polymorphic enzymes that can cause abolished, quantitatively or qualitatively altered, or enhanced drug metabolism. Ultrarapid metabolism is due to stable duplication, multiduplication, or amplification of active genes. Several examples exist where subjects carrying certain alleles suffer from a lack of drug efficacy due to ultrarapid metabolism or, alternatively, adverse effects from the drug treatment due to the presence of defective alleles. The polymorphic enzymes create a problem for the drug industry because of the extensive interindividual variability in the metabolism of candidate drugs that are substrates for such enzymes. The new area for lead generation has a more preclinical emphasis and involves combinatorial chemistry in conjunction with high-throughput-based analysis of thousands of substances with respect to their absorption, metabolism, and excretion characteristics. The outcome is that companies drop substrates for polymorphic enzymes at an early stage in development, which will of course create fewer problems with polymorphic enzymes in the future. The risk is that very valuable candidates, which cannot be replaced easily, never come out on the market. The alternative, however, of using the patient's genotype as a basis for individualized drug treatment constitutes, in light of rapid methodological developments, a very feasible approach to safer and more efficient drug therapies.

Cytochrome P-450 Enzyme System↗
PubMed2001

Transcriptional and post-translational regulation of CYP1A1 by primaquine.

Regulation of the CYP1A1 gene has been shown to involve the aryl hydrocarbon receptor and the CYP1A1 gene expression is induced by AhR ligands. Primaquine is an antimalarial agent that does not exhibit the structural properties of a classical AhR ligand. We have evaluated the mechanisms by which this compound induces CYP1A1 expression using rat hepatoma H4IIE cells and V79 cells stably expressing CYP1A1. In H4IIE cells, primaquine caused a time- and dose-dependent increase of CYP1A1 mRNA and protein expression. The transcriptional activation of the CYP1A1 gene by primaquine was strictly XRE-dependent, as shown by transfection of different CYP1A1 pGL3 reporter constructs in H4IIE cells, and the involvement of the AhR was shown by activation of a Gal4-AhR hybrid protein by primaquine in transfected cells. Furthermore, primaquine caused transformation of the cytosolic AhR to a DNA-binding form, in vitro, suggesting that primaquine directly activates the receptor complex. In addition to its action at the transcriptional level, primaquine caused a dose-dependent inhibition of CYP1A1 degradation with an IC(50) of 3.3 , as seen in mammalian V79 cells. This was not due to the lysosomotropic activity of the drug since other lysosomotropic agents were ineffective. Primaquine formed a type II binding spectrum with CYP1A1 and inhibited the CYP1A1-dependent ethoxyresorufin O-deethylase activity in vitro with a K(i) of 1.3 microM, which is close to the IC(50), suggesting that the drug protects CYP1A1 from degradation by binding at the active site. It is concluded that CYP1A1 is regulated by primaquine both on the transcriptional as well as on a post-translational level.

Animals↗
PubMed2001

An assessment of human liver-derived in vitro systems to predict the in vivo metabolism and clearance of almokalant.

The ability of various human derived in vitro systems to predict various aspects of the in vivo metabolism and kinetics of almokalant have been investigated in a multicenter collaborative study. Although almokalant has been withdrawn from further clinical development, its metabolic and pharmacokinetic properties have been well characterized. Studies with precision-cut liver slices, primary hepatocyte cultures, and hepatic microsomal fractions fortified with UDP-glucuronic acid all suggested that almokalant is mainly glucuronidated to the stereoisomers M18a and M18b, which is in good agreement with the results in vivo. Both in vivo and in vitro studies indicate that the formation of M18b dominates over that of M18a, although the difference is more pronounced with the in vitro systems. Molecular modeling, cDNA-expressed enzyme analysis, correlation analysis, and inhibition studies did not clearly indicate which P450 enzymes catalyze the oxidative pathways, which may indicate a problem in identifying responsible enzymes for minor metabolic routes by in vitro methods. All of the in vitro systems underpredicted the metabolic clearance of almokalant, which has previously been reported to be a general problem for drugs that are cleared by P450-dependent metabolism. Although few studies on in vivo prediction of primarily glucuronidated drugs have appeared, in vitro models may consistently underpredict in vivo metabolic clearance. We conclude that in vitro systems, which monitor phase II metabolism, would be beneficial for prediction of the in vivo metabolism, although all of the candidate liver-derived systems studied here, within their intrinsic limitations, provided useful information for predicting metabolic routes and rates.

Anti-Arrhythmia Agents↗
PubMed2001

Role of CYP2C9 polymorphism in losartan oxidation.

Losartan, an angiotensin II receptor antagonist, is oxidized by hepatic cytochromes P450 to an active carboxylic acid metabolite, E-3174. The aim of the present investigation was to study the contribution of CYP2C9 and CYP3A4 in losartan oxidation in vitro and to evaluate the role of CYP2C9 polymorphism. Kinetic properties of different genetic CYP2C9 variants were compared both in a yeast expression system and in 25 different samples of human liver microsomes where all known genotypes of CYP2C9 were represented. Microsomes were incubated with losartan (0.05-50 microM), and the formation of E-3174 was analyzed by high-performance liquid chromatography to estimate V(max), K(m), and intrinsic clearance for all individual samples. Sulfaphenazole, a CYP2C9 inhibitor, blocked the formation of E-3174 at low losartan concentrations (<1 microM), whereas the inhibitory effect of triacetyloleandomycin, a CYP3A4 inhibitor, was significant only at high concentrations of losartan (>25 microM). In comparison to the CYP2C9.1 variant, oxidation of losartan was significantly reduced in yeast expressing the rare CYP2C9.2 or CYP2C9.3 variants. Moreover, the rate of losartan oxidation was lower in liver microsomes from individuals hetero- or homozygous for the CYP2C9*3 allele, or homozygous for the CYP2C9*2 allele. The difference between the common and rare CYP2C9 variants was mainly explained by a lower V(max), both in yeast and human liver microsomes. In summary, these in vitro results indicate that CYP2C9 is the major human P450 isoenzyme responsible for losartan oxidation and that the CYP2C9 genotype contributes to interindividual differences in losartan oxidation and activation.

Angiotensin Receptor Antagonists↗
PubMed2001

CYP2B6 and CYP2C19 as the major enzymes responsible for the metabolism of selegiline, a drug used in the treatment of Parkinson's disease, as revealed from experiments with recombinant enzymes.

In view of conflicting data in the literature regarding the enzyme(s) responsible for metabolism of selegiline, a drug used in the treatment of Parkinson's disease, investigations were carried out in vitro using the human cytochrome P450 enzymes CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 recombinantly expressed in yeast to elucidate the enzyme specificity in selegiline metabolism. In the yeast microsomes used, desmethylselegiline and levomethamphetamine were formed from selegiline at significant rates. The highest contribution to the hepatic clearance of selegiline was calculated to be exerted by CYP2B6 (124 l/h) CYP2C19 (82 l/h), whereas CYP3A4 (27 l/h) and CYP1A2 (21 l/h) were of less importance. Antibodies against CYP2B6 inhibited metabolism of selegiline in microsomes containing CYP2B6 but not in microsomes without significant amounts of the enzyme. In contrast to previous reports, we could not find any role for CYP2D6 in the metabolism of selegiline. The data strongly indicate that the high extent of interindividual variation seen in vivo for selegiline clearance is caused by the metabolism of the compound by the highly polymorphic CYP2B6 and CYP2C19.

Antiparkinson Agents↗
PubMed2001

Metabolic gene polymorphism frequencies in control populations.

Using the International Project on Genetic Susceptibility to Environmental Carcinogens (GSEC) database containing information on over 15,000 control (noncancer) subjects, the allele and genotype frequencies for many of the more commonly studied metabolic genes (CYP1A1, CYP2E1, CYP2D6, GSTM1, GSTT1, NAT2, GSTP, and EPHX) in the human population were determined. Major and significant differences in these frequencies were observed between Caucasians (n = 12,525), Asians (n = 2,136), and Africans and African Americans (n = 996), and some, but much less, heterogeneity was observed within Caucasian populations from different countries. No differences in allele frequencies were seen by age, sex, or type of controls (hospital patients versus population controls). No examples of linkage disequilibrium between the different loci were detected based on comparison of observed and expected frequencies for combinations of specific alleles.

Black People↗
PubMed2000

Identification and characterization of a mitochondrial targeting signal in rat cytochrome P450 2E1 (CYP2E1).

Cytochrome P450 2E1 (CYP2E1) lacking the hydrophobic NH(2)-terminal hydrophobic transmembrane domain is specifically targeted to mitochondria, where it is processed to a soluble and catalytically active form (Delta2E1) with a mass of about 40 kDa. Small amounts of Delta2E1 were also observed in mitochondria isolated from rat liver, indicating that this form of CYP2E1 is also present in vivo. In the present study the mitochondrial targeting signal was identified and characterized by the use of several NH(2)-terminally truncated and mutated forms of CYP2E1 that were expressed in the mouse H2.35 hepatoma cell line. Two potential mitochondrial targeting sequences were identified in the NH(2) terminus of CYP2E1. Deletion of the first potential mitochondrial targeting sequence located between amino acids 50 and 65, as in Delta(2-64)2E1, still resulted in mitochondrial targeting and processing, but when, in addition to the first, the second potential mitochondrial targeting sequence located between amino acids 74 and 95 was also deleted, as in Delta(2-95)2E1, the mitochondrial targeting was abolished. Mutation of the four positively charged Arg and Lys residues present in this sequence to neutral Ala residues resulted in the abrogation of mitochondrial targeting. Deletion of a hydrophobic stretch of amino acids between residues 76 and 83 also abolished mitochondrial targeting and import. Once imported in the mitochondria, these constructs were further processed to the mature protein Delta2E1. It is concluded that mitochondrial targeting of CYP2E1 is mediated through a sequence located between residues 74 and 95 and that positively charged residues as well as a hydrophobic stretch present in the beginning of this sequence are essential for this process.

Amino Acid Sequence↗
PubMed2000

The effect of ethanol-induced cytochrome p4502E1 on the inhibition of proteasome activity by alcohol.

The present investigation was undertaken to determine the effect of CYP2E1 induction by ethanol on the inhibition of proteasomal activity in wild-type and CYP2E1 knockout C57 black mice. The proteasomal chymotrypsin-like activity decreased significantly in ethanol-fed wild-type mice liver, but was not reduced in ethanol-fed knockout mice liver. The 26S proteasomal activity was decreased more by ethanol feeding than was the 20S proteasomal fraction. Individual hepatocytes lost immunostaining of the proteasomes in the centrilobular zone in the livers of ethanol-fed wild-type mice and the knockout mouse liver. There was increased product of protein oxidation in the liver in the wild type but not in the knockout mice given ethanol. Taken together, these results suggest that CYP2E1 induction was responsible for the decrease in proteasome activity seen in the wild-type mice which head to the accumulation of oxidized proteins which were increased as the result of free radicals generated by CYP2E1 metabolism of ethanol.

Animals↗
PubMed2000

Cyclic expression of class I alcohol dehydrogenase in male rats treated with ethanol.

Continuous infusion of ethanol-containing diets has been demonstrated to generate well-defined pulses in blood and urine ethanol concentrations that occur with a frequency of approximately 6 days. The present study aimed to determine if hepatic class I alcohol dehydrogenase was the cause of these cycles. Adult male rats were fed an ethanol-containing diet by continuous intragastric infusion. Hepatic ADH activity, class I ADH mRNA level and rate of class I ADH gene transcription fluctuated in a cyclic pattern that positively correlated with UECs, and inhibition of ADH with 4-methylpyrazole abolished the UEC pulses. These data demonstrate for the first time an ethanol-dependent regulation of rat hepatic class I ADH. The cyclic behavior of the ethanol levels correlates with changes in class I ADH expression and implies adaptability of the ethanol eliminating system to high concentrations of alcohol.

Alcohol Dehydrogenase↗
PubMed2000

Identification of CYP4F8 in human seminal vesicles as a prominent 19-hydroxylase of prostaglandin endoperoxides.

A novel cytochrome P450, CYP4F8, was recently cloned from human seminal vesicles. CYP4F8 was expressed in yeast. Recombinant CYP4F8 oxygenated arachidonic acid to (18R)-hydroxyarachidonate, whereas prostaglandin (PG) D(2), PGE(1), PGE(2), PGF(2alpha), and leukotriene B(4) appeared to be poor substrates. Three stable PGH(2) analogues, 9,11-epoxymethano-PGH(2) (U-44069), 11, 9-epoxymethano-PGH(2) (U-46619), and 9,11-diazo-15-deoxy-PGH(2) (U-51605) were rapidly metabolized by omega2- and omega3-hydroxylation. U-44069 was oxygenated with a V(max) of approximately 260 pmol min(-)(1) pmol P450(-1) and a K(m) of approximately 7 micrometer. PGH(2) decomposes mainly to PGE(2) in buffer and to PGF(2alpha) by reduction with SnCl(2). CYP4F8 metabolized PGH(2) to 19-hydroxy-PGH(2), which decomposed to 19-hydroxy-PGE(2) in buffer and could be reduced to 19-hydroxy-PGF(2alpha) with SnCl(2). 18-Hydroxy metabolites were also formed (approximately 17%). PGH(1) was metabolized to 19- and 18-hydroxy-PGH(1) in the same way. Microsomes of human seminal vesicles oxygenated arachidonate, U-44069, U-46619, U-51605, and PGH(2), similar to CYP4F8. (19R)-Hydroxy-PGE(1) and (19R)-hydroxy-PGE(2) are the main prostaglandins of human seminal fluid. We propose that they are formed by CYP4F8-catalyzed omega2-hydroxylation of PGH(1) and PGH(2) in the seminal vesicles and isomerization to (19R)-hydroxy-PGE by PGE synthase. CYP4F8 is the first described hydroxylase with specificity and catalytic competence for prostaglandin endoperoxides.

Aryl Hydrocarbon Hydroxylases↗
PubMed2000

Molecular basis for the transport of cytochrome P450 2E1 to the plasma membrane.

Endoplasmic reticulum-resident cytochrome P450 enzymes that face the cytosol are present on the plasma membrane of hepatocytes, but the molecular origin for their transport to this compartment has until now remained unknown. The molecular basis for the transport of rat ethanol-inducible cytochrome P450 2E1 (CYP2E1) to the plasma membrane was investigated by transfection of several different mutant cDNAs into mouse H2.35 hepatoma cells. Two NH(2)-terminal CYP2E1 mutants were constructed: N(++)2E1, which carried two positive charges in the NH(2) terminus, and 2C-2E1, in which the transmembrane domain of CYP2E1 was replaced with that of CYP2C1, which was previously described to cause retention of CYP2C1 in the endoplasmic reticulum, as well as CYP2E1 COOH-terminally tagged with the vesicular stomatitis virus G protein (VSV-G) epitope (2E1-VSV-G). Immunofluorescent microscopy and cell surface biotinylation experiments revealed that all CYP2E1 variants were present on the extracellular side of the plasma membrane. The VSV-G epitope on CYP2E1 was detected on the outside of the plasma membrane using VSV-G-specific antibodies, indicating that the large COOH-terminal part of CYP2E1 is indeed exposed on the outside of the plasma membrane. The relative levels of CYP2E1, 2C-2E1, and 2E1-VSV-G on the cell surface were found to be about 2% of total cellular enzyme, whereas twice this amount of N(++)2E1 was recovered at the cell surface. Protease protection experiments performed on microsomes isolated from cDNA transfected cells revealed that a small fraction of CYP2E1 and all variant proteins was found to be located in the lumen of the endoplasmic reticulum (type II orientation), whereas the majority of the proteins were in the expected cytosolic or type I orientation. It is concluded that the NH(2)-terminal transmembrane domain of CYP2E1 plays a critical role in directing the protein to the cell surface and that topological inversion of a small fraction of CYP2E1 in the endoplasmic reticulum directs the protein to the plasma membrane.

Animals↗
PubMed2000

Characterization and functional analysis of two common human cytochrome P450 1B1 variants.

Cytochrome P450 1B1 (CYP1B1) is a human extrahepatic P450 that activates procarinogens, metabolizes 17beta-estradiol, and may well have a role in the pathogenesis of some forms of cancer. Besides rare deleterious mutations reported for the CYP1B1 gene, six single-nucleotide polymorphisms have been reported, of which four cause amino acid exchanges. We have expressed two of the common CYP1B1 alleles in yeast cells and mammalian COS-1 cells in order to functionally characterize the alleles with respect to kinetic properties and protein stability. The CYP1B1.2 variant contains the two linked amino acid substitutions R48G and A119S compared to CYP1B1.1. The kinetic parameters of two structurally unrelated CYP1B1 substrates for the two variants were examined. No kinetic differences were seen of 17beta-estradiol hydroxylation activities between the two CYP1B1 variants and an only minor increase in the apparent Km for ethoxyresorufin was observed for CYP1B1.2. It therefore appears that they have very similar catalytic properties and the substitutions do not appear to alter CYP1B1 catalytic function. The two CYP1B1 variants were similarly stable when expressed in mammalian COS-1 cells, indicating that the substitutions have no effect on protein folding or stability. The combined results indicate that these two CYP1B1 variants show very similar properties with respect to catalytic activities and protein stability and do not alter CYP1B1 function.

Amino Acid Sequence↗