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M Ingelman-Sundberg

Publications and source records attributed to M Ingelman-Sundberg.

At least 19 recordsLinked to original sources
PubMed2006

Cytochrome P450 pharmacogenetics and cancer.

The cytochromes P450 (CYPs) are key enzymes in cancer formation and cancer treatment. They mediate the metabolic activation of numerous precarcinogens and participate in the inactivation and activation of anticancer drugs. Since all CYPs that metabolize xenobiotics are polymorphic, much emphasis has been put on the investigation of a relationship between the distribution of specific variant CYP alleles and risk for different types of cancer, but a consistent view does not yet exist. This is to a great extent explained by the fact that the CYPs involved in activation of precarcinogens are in general not functionally polymorphic. This is in contrast to CYPs that are active in drug biotransformation where large interindividual differences in the capacity to metabolize therapeutic drugs are seen as a consequence of polymorphic alleles with altered function. This includes also some anticancer drugs like tamoxifen and cyclophosphamide metabolized by CYP2D6, CYP2C19 and CYP2B6. Some P450 forms are also selectively expressed in tumours, and this could provide a mechanism for drug resistance, but also future therapies using these enzymes as drug targets can be envisioned. This review gives an up-to-date description of our current knowledge in these areas.

Antineoplastic Agents↗
PubMed2005

The role of phenylalanine 483 in cytochrome P450 2D6 is strongly substrate dependent.

The polymorphic cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 30% of the drugs currently prescribed, and is thus clinically relevant. Typical CYP2D6 substrates generally contain a basic nitrogen atom and an aromatic moiety adjacent to the site of metabolism. Recently, we demonstrated the importance of active site residue F120 in substrate binding and catalysis in CYP2D6. On the basis of protein homology models, it is claimed that another active site phenylalanine, F483, may also play an important role in the interaction with the aromatic moiety of CYP2D6 substrates. Experimental data to support this hypothesis, however, is not yet available. In fact, in the only study performed, mutation of F483 to isoleucine or tryptophan did not affect the 1'-hydroxylation of bufuralol at all [Smith G, Modi S, Pillai I, Lian LY, Sutcliffe MJ, Pritchard MP, et al., Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity. Biochem J 1998;331:783-92]. In the present study, the role of F483 in ligand binding and metabolism by CYP2D6 was examined experimentally using site-directed mutagenesis. Replacement of F483 by alanine resulted in a 30-fold lower V(max) for bufuralol 1'-hydroxylation, while the K(m) was hardly affected. The V(max) for 3,4-methylenedioxy-methylamphetamine O-demethylenation on the other hand decreased only two-fold, whereas the effect on the K(m) was much larger. For dextromethorphan, in addition to dextrorphan (O-demethylation) and 3-methoxymorphinan (N-demethylation), two other metabolites were formed that could not be detected for the wild-type. The substrate 7-methoxy-4-(aminomethyl)-coumarin was not metabolised at all by CYP2D6[F483A], a phenomenon that was reported also for CYP2D6[F120A]. The presented data show that next to F120, residue F483 plays a very important role in the metabolism of typical CYP2D6 substrates. The influence of F483 on metabolism was found to be strongly substrate-dependent.

Cytochrome P-450 CYP2D6↗
PubMed2005

Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity.

CYP2D6 is of great importance for the metabolism of clinically used drugs and about 20-25% of those are metabolised by this enzyme. In addition, the enzyme utilises hydroxytryptamines as endogenous substrates. The polymorphism of the enzyme results in poor, intermediate, efficient or ultrarapid metabolisers (UMs) of CYP2D6 drugs. It is plausible that the UM genotype, where more than one active gene on one allele occurs, is the outcome of selective dietary selection in certain populations in North East Africa. The UM phenotype affects 5.5% of the population in Western Europe. A hypothesis for the evolutionary basis behind selection for CYP2D6 gene duplications is presented in relation to selection for Cyp6 variants in insecticide resistant Drosophila strains. The polymorphism of CYP2D6 significantly affects the pharmacokinetics of about 50% of the drugs in clinical use, which are CYP2D6 substrates. The consequences of the polymorphism at ordinary drug doses can be either adverse drug reactions or no drug response. Examples are presented where CYP2D6 polymorphism affects the efficacy and costs of drug treatment. Predictive CYP2D6 genotyping is estimated by the author to be beneficial for treatment of about 30-40% of CYP2D6 drug substrates, that is, for about 7-10% of all drugs clinically used, although prospective clinical studies are necessary to evaluate the exact benefit of drug selection and dosage based on the CYP2D6 genotype.

Animals↗
PubMed2003

Transcriptional regulation of human CYP3A4 basal expression by CCAAT enhancer-binding protein alpha and hepatocyte nuclear factor-3 gamma.

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more than 50% of currently used therapeutic drugs, yet the mechanisms that control CYP3A4 basal expression in liver are poorly understood. Several putative binding sites for CCAAT/enhancer-binding protein (C/EBP) and hepatic nuclear factor 3 (HNF-3) were found by computer analysis in CYP3A4 promoter. The use of reporter gene assays, electrophoretic mobility shift assays, and site-directed mutagenesis revealed that one proximal and two distal C/EBP alpha binding sites are essential sites for the trans-activation of CYP3A4 promoter. No trans-activation was found in similar reporter gene experiments with a HNF-3 gamma expression vector. The relevance of these findings was further explored in the more complex DNA/chromatin structure within endogenous CYP3A4 gene. Using appropriate adenoviral expression vectors, we found that both hepatic and nonhepatic cells overexpressing C/EBP alpha had increased CYP3A4 mRNA levels, but no effect was observed when HNF-3 gamma was overexpressed. In contrast, overexpression of HNF-3 gamma simultaneously with C/EBP alpha resulted in a greater activation of the CYP3A4 gene. This cooperative effect was hepatic-specific and also occurred in CYP3A5 and CYP3A7 genes. To investigate the mechanism for HNF-3 gamma action, we studied its binding to CYP3A4 promoter and the effect of the deacetylase inhibitor trichostatin A. HNF-3 gamma was able to bind CYP3A4 promoter at a distal position, near the most distal C/EBP alpha binding site. Trichostatin A increased C/EBP alpha effect but abolished HNF-3 gamma cooperative action. These findings revealed that C/EBP alpha and HNF-3 gamma cooperatively regulate CYP3A4 expression in hepatic cells by a mechanism that probably involves chromatin remodeling.

Adenoviridae↗
PubMed2002

Autoantibodies against cytochrome P450s in sera of children treated with immunosuppressive drugs.

Treatment with the immunosuppressive drugs cyclosporin and tacrolimus, the mainstays of anti-graft rejection and autoimmune disease therapy, is limited by their hepato- and nephrotoxicity. The metabolic conversion of these compounds to more easily excretable products is catalysed mainly by hepatic cytochrome P4503A4 (CYP3A4) but also involves extrahepatic CYP3A5 and other P450 forms. We set out to study whether or not exposure to cyclosporin and FK506 in children undergoing organ transplantation leads to formation of autoantibodies against P450s. Immunoblotting analysis revealed anti-CYP reactivity in 16% of children on CyA for anti-graft rejection or treatment of nephrosis (n = 67), 31% of kidney transplant patients switched from CyA to FK506 (n = 16), and 21% of kidney and or liver transplant patients on FK506 (n = 14). In contrast, the frequency of reactive immunoblots was only 8.5% among the normal paediatric controls (n = 25) and 7% among adult kidney transplant patients on CyA or FK506 (n = 30). The CYP2C9+ sera were able to immunoprecipitate in vitro translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti-cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection.

Adolescent↗
PubMed2001

The importance of residues in substrate recognition site 3 for the catalytic function of CYP2D25 (vitamin D 25-hydroxylase).

Porcine CYP2D25, microsomal vitamin D(3) 25-hydroxylase, catalyzes the essential first step in the bioactivation of the prohormone vitamin D(3). Although CYP2D25 shows a high degree of sequence identity with other members of the CYP2D subfamily, such as human CYP2D6, the vitamin D(3) 25-hydroxylase activity is a unique property among CYP2D enzymes. In addition to 25-hydroxylation, CYP2D25 also metabolizes the drug tolterodine. In this study, CYP2D25 was functionally expressed in the Saccharomyces cerevisiae W(R) strain and site-directed mutagenesis was used to study the role of substrate recognition site 3 (SRS-3) for the catalytic specificity of CYP2D25. Five residues in SRS-3 of CYP2D25 were simultaneously mutated to the equivalent residues in CYP2D6, an enzyme not active in 25-hydroxylation. Western blot analysis of microsomes from transformed yeast cells showed that both the wild-type and mutant CYP2D25 were expressed at comparable levels. The 25-hydroxylase activity of recombinant mutant CYP2D25 was completely lost whereas the activity toward tolterodine remained virtually unaffected. The results implicate that residues in SRS-3 of CYP2D25 are important determinants for its function in vitamin D(3) metabolism.

Amino Acid Sequence↗
PubMed2001

Genetic susceptibility to adverse effects of drugs and environmental toxicants. The role of the CYP family of enzymes.

The majority of cytochrome P450 (CYP)-dependent xenobiotic metabolism is carried out by polymorphic and inducible enzymes which can cause abolished, quantitatively or qualitatively altered or enhanced drug metabolism. Stable duplication, multi-duplication or amplification of active genes, most likely in response to dietary components causing a selection of alleles with multiple genes, has been described. Several examples exist where subjects carrying certain alleles suffer from a lack of drug efficacy due to ultra-rapid metabolism caused by multiple genes or by induction of gene expression or, alternatively, adverse effects from the drug treatment due to the presence of defective alleles. The polymorphism of CYP enzymes is expected to influence the individual sensitivity and toxicity for different environmental agents, although there is no real consensus in the literature about specific firm relationships in this regard. Dosage requirements for several commonly used drugs that have a narrow therapeutic range can differ more than 20-fold dependent on the genotype or the enzyme expression status. The incidence of serious and fatal adverse drug reactions has been found to be very high among hospitalised patients and causes over 100,000 deaths per year in the US, making it between the 4th and 6th leading cause of death. It is likely that predictive genotyping could avoid 10-20% of these deaths. In the present contribution, an overview is presented about our present knowledge about the polymorphism of xenobiotic metabolising CYPs and the importance for adverse effects of drugs and metabolic activation of xenobiotics.

Cytochrome P-450 Enzyme System↗
PubMed2001

Identification of a single nucleotide polymorphism in the TATA box of the CYP2A6 gene: impairment of its promoter activity.

Human cytochrome P450 2A6 (CYP2A6) constitutes the major nicotine oxidase, and large interindividual differences are seen in the levels of this enzyme, to a great extent caused by the distribution of several different polymorphic gene variants mainly located in the open reading frame (ORF). In the present study, we report a common polymorphism located in the 5' flanking region of CYP2A6 affecting its expression. DHPLC analysis and complete sequence of the open reading frame of the gene from a Turkish individual revealed a -48T > G substitution disrupting the TATA box. Using dynamic allele-specific hybridization (DASH), genotyping of this novel variant (named CYP2A6*9) was carried out in 116 Swedish, 132 Turkish, and 102 Chinese subjects, and the allele frequencies were found to be 5.2, 7.2, and 15.7%, respectively. The significance of the polymorphism was investigated by the construction of luciferase reporter plasmids containing 135 or 500 bp of the 5'-upstream region of the gene transfected into human hepatoma B16A2 cells. The constructs carrying the -48T > G mutation were only expressed at about 50% of the wild-type alleles. It is concluded that the CYP2A6*9 allele might be one of the most common CYP2A6 variants in Caucasians that alters the levels of enzyme expression.

Alleles↗
PubMed2001

Genetic variability in susceptibility and response to toxicants.

Xenobiotic metabolism is carried out by phase I and phase II enzymes which are to a large extent polymorphic. The majority of cytochrome P450 (CYP) enzymes involved in xenobiotic metabolism are polymorphic and inducible, resulting in abolished, quantitatively or qualitatively altered or enhanced drug metabolising activity. Stable duplication, multiduplication or amplification of active genes have been described. In mouse models it is apparent that inactivation of specific enzymes active in xenobiotic metabolism can affect the risk for cancer development in relation to specific xenobiotic exposure, whereas the situation in humans is far more complex. The polymorphism of CYP enzymes is expected to influence individual sensitivity and toxicity for different environmental agents, although there is as yet no real consensus in the literature about specific firm relationships in this regard. The incidence of serious and fatal adverse drug reactions (ADRs) has been found to be very high among hospitalised patients, the cost of ADRs to society is large and they are responsible for 5-10% of all hospital admissions. It is likely that predictive genotyping could avoid 10-20% of ADRs. In the present contribution an overview is presented regarding our present knowledge about the polymorphism of phase I enzymes, with emphasis on xenobiotic metabolising CYPs and the importance for metabolic activation of xenobiotics.

Cytochrome P-450 Enzyme System↗
PubMed2001

Identification and tissue distribution of the novel human cytochrome P450 2S1 (CYP2S1).

With the aid of the htgs and dbEST databases, a novel cytochrome P450 cDNA was found by homology searches, and the corresponding gene was identified on chromosome 19. Nested PCR was used to amplify a full-length sequence of 1515 bp. The predicted 504 amino acid sequence displays 38--49% identity with CYP2 family members and the protein was designated CYP2S1. mRNA dot blot analysis demonstrated high expression levels in trachea, lung, stomach, small intestine, and spleen. The expression pattern was confirmed by Northern blot, which also revealed a single transcript of approximately 2.4 kb. Western blot analysis, using an antiserum directed against the C-terminus of the enzyme, detected a protein in human lung with the same mobility as recombinant CYP2S1. Subcellular fractionation and immunostaining revealed that CYP2S1 was localized in the endoplasmic reticulum. We conclude that CYP2S1 represents a novel abundantly expressed human P450 with potential importance for extrahepatic xenobiotic metabolism.

Amino Acid Sequence↗
PubMed2001

Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43.

On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism.

Amino Acid Sequence↗
PubMed2001

Thioredoxin fold as homodimerization module in the putative chaperone ERp29: NMR structures of the domains and experimental model of the 51 kDa dimer.

BACKGROUND: ERp29 is a ubiquitously expressed rat endoplasmic reticulum (ER) protein conserved in mammalian species. Fold predictions suggest the presence of a thioredoxin-like domain homologous to the a domain of human protein disulfide isomerase (PDI) and a helical domain similar to the C-terminal domain of P5-like PDIs. As ERp29 lacks the double-cysteine motif essential for PDI redox activity, it is suggested to play a role in protein maturation and/or secretion related to the chaperone function of PDI. ERp29 self-associates into 51 kDa dimers and also higher oligomers. RESULTS: 3D structures of the N- and C-terminal domains determined by NMR spectroscopy confirmed the thioredoxin fold for the N-terminal domain and yielded a novel all-helical fold for the C-terminal domain. Studies of the full-length protein revealed a short, flexible linker between the two domains, homodimerization by the N-terminal domain, and the presence of interaction sites for the formation of higher molecular weight oligomers. A gadolinium-based relaxation agent is shown to present a sensitive tool for the identification of macromolecular interfaces by NMR. CONCLUSIONS: ERp29 is the first eukaryotic PDI-related protein for which the structures of all domains have been determined. Furthermore, an experimental model of the full-length protein and its association states was established. It is the first example of a protein where the thioredoxin fold was found to act as a specific homodimerization module, without covalent linkages or supporting interactions by further domains. A homodimerization module similar as in ERp29 may also be present in homodimeric human PDI.

Amino Acid Sequence↗
PubMed2001

Genetic polymorphism of cytochrome P450 2C9 in a Caucasian and a black African population.

AIMS: CYP2C9 is a major enzyme in human drug metabolism and the polymorphism observed in the corresponding gene may affect the therapeutic outcome during treatment with several drugs. The distribution of variant CYP2C9 alleles was therefore investigated in an Italian and an Ethiopian population. METHODS: Allele-specific PCR analysis was carried out in order to determine the frequencies of the two most common variant alleles, CYP2C9*2 and CYP2C9*3 in genomic DNA isolated from 157 Italians and 150 Ethiopians. RESULTS: The frequencies of CYP2C9*1 (80%), CYP2C9*2 (11%) and CYP2C9*3 (9%) found in the Italian population were similar to other Caucasian groups. However in the Ethiopian population CYP2C9*1, CYP2C9*2 and CYP2C9*3 were present at a frequency of 94, 4 and 2% respectively. The 95% confidence intervals in CYP2C9*1, CYP2C9*2 and CYP2C9*3 between Italians and Ethiopians were 0.098, 0.176, 0.040, 0.098 and 0.040, 0.098, respectively. CONCLUSIONS: Our results indicate that the Ethiopian population has a unique relative distribution of the CYP2C9 alleles, which is not similar to any other ethnic group hitherto described.

Adult↗
PubMed2001

Pharmacogenetics: an opportunity for a safer and more efficient pharmacotherapy.

Drug treatment is in many cases ineffective. Besides patients who do not respond to the treatment despite receiving expensive drugs, adverse drug reactions (ADRs) as a consequence of the treatment, is estimated to cost the US society 100 billion USD and over 100,000 deaths per year. Pharmacogenetics is the discipline which takes the patient's genetic information of drug transporters, drug metabolizing enzymes and drug receptors into account to allow for an individualized drug therapy leading to optimal choice and dose of the drugs in question. It is believed that much cost for the society can be saved in this manner. Many drug transporters are polymorphic. In addition, the majority of phase I and phase II dependent drug metabolism is carried out by polymorphic enzymes which can cause abolished, quantitatively or qualitatively altered or enhanced drug metabolism. Stable duplication, multiduplication or amplification of active genes, most likely in response to dietary components that have resulted in a selection of alleles with multiple noninducible genes, has been described. Several examples exist where subjects carrying certain alleles suffer from a lack of drug efficacy because of ultrarapid metabolism caused by multiple genes or by induction of gene expression, or, alternatively, adverse effects from the drug treatment as a result of the presence of defective alleles. The information about the role of polymorphic drug receptors for efficiency of drug therapy is more scarce, although promising examples are seen in drug treatment of asthma where the efficiency can be severely enhanced by predictive genotyping of the drug targets. In addition, certain polymorphic genes can be used as markers for optimization of the drug therapy. It is likely that predictive genotyping is of benefit in 10-20% of drug treatment and thereby allows for prevention of causalities as a cause of ADRs and thus improves the health for a significant fraction of the patients. In 15-40% of the cases, the penetrance of genetic polymorphism is of less importance because of the polygenic influence on the outcome of drug treatment and in 50% of the cases, pharmacogenetics would be without influence because of other more important physiological and environmental factors. In the present contribution an overview about our present knowledge how polymorphic genes can influence the drug efficacy is presented. Some emphasis will be given to different forms of cytochrome P450 which are of importance for drug metabolism.

Cytochrome P-450 Enzyme System↗
PubMed2001

Carbamazepine: a 'blind' assessment of CVP-associated metabolism and interactions in human liver-derived in vitro systems.

1. The ability of various in vitro systems for CYP enzymes (computer modelling, human liver microsomes, precision-cut liver slices, hepatocytes in culture, recombinant enzymes) to predict various aspects of in vivo metabolism and kinetics of carbamazepine (CBZ) was investigated. 2. The study was part of the EUROCYP project that aimed to evaluate relevant human in vitro systems to study drug metabolism. 3. CBZ was given to the participating laboratories without disclosing its chemical nature. 4. The most important enzyme (CYP3A4) and metabolic route (10,11-epoxidation) were predicted by all the systems studied. 5. Minor enzymes and routes were predicted to a different extent by various systems. 6. Prediction of a clearance class, i.e. slow clearance, was correctly predicted by microsomes, slices, hepatocytes and recombinant enzymes (CYP3A4). 7. The 10,11-epoxidation of CBZ by the recombinant CYP3A4 was enhanced by the addition of exogenous cytochrome-b5, leading to a considerable over-prediction. 8. Induction potency of CBZ was predicted in cultured hepatocytes in which 7-ethoxycoumarin O-deethylase was used as an index activity. 9. It seems that for a principally CYP-metabolized substance such as CBZ, all liver-derived systems provide useful information for prediction of metabolic routes, rates and interactions.

Anticonvulsants↗
PubMed2001

The ubiquitin-proteasome 26s pathway in liver cell protein turnover: effect of ethanol and drugs.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Samuel W. French and R. J. Mayer. The presentations were (1) The ubiquitin-proteasome 26s pathway in liver cell protein turnover: Effect of alcohol and drugs, by Samuel W. French and F. Bardag-Gorce; (2) The role of CYP2E1 phosphorylation and degradation pathway in the induction of the enzyme, by Magnus Ingelman-Sundberg; (3) Role of proteasome in the proteolysis of oxidized proteins in experimental chronic alcoholism, by Helen Rouach; (4) Alcohol, proteolysis and liver cancer, by R. J. Mayer; (5) Effect of ethanol feeding on the ATP-ubiquitin-proteasome pathway in the liver cell, by F. Bardag-Gorce; (6) Novel mechanisms and targets for intracellular transport of CYP2E1, by E. Neve; and (7) Gankyrin, an oncoprotein commonly over expressed in hepatoma, by H. Higashitsuji.

Animals↗
PubMed2001

Ethanol and oxidative stress.

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was Albert Y. Sun. The presentations were (1) Ethanol-inducible cytochrome P-4502E1 in alcoholic liver disease, by Magnus Ingelman-Sundberg and Etienne Neve; (2) Regulation of NF-kappaB by ethanol, by H. Matsumoto, Y. Nishitani, Y. Minowa, and Y. Fukui; (3) Chronic ethanol consumption increases concentration of oxidized proteins in rat liver, by Shannon M. Bailey, Vinood B. Patel, and Carol C. Cunningham; (4) Antiphospholipids antibodies and oxidized modified low-density lipoprotein in chronic alcoholic patients, by Tomas Zima, Lenka Fialova, Ludmila Mikulikova, Ptr Popov, Ivan Malbohan, Marta Janebova, and Karel Nespor; and (5) Amelioration of ethanol-induced damage by polyphenols, by Albert Y. Sun and Grace Y. Sun.

Alcoholism↗