Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo.

To evaluate whether alterations in the multidrug-resistance (MDR)-1 gene correlate with intestinal MDR-1 expression and uptake of orally administered P-glycoprotein (PGP) substrates, we analyzed the MDR-1 sequence in 21 volunteers whose PGP expression and function in the duodenum had been determined by Western blots and quantitative immunohistology (n = 21) or by plasma concentrations after orally administered digoxin (n = 8 + 14). We observed a significant correlation of a polymorphism in exon 26 (C3435T) of MDR-1 with expression levels and function of MDR-1. Individuals homozygous for this polymorphism had significantly lower duodenal MDR-1 expression and the highest digoxin plasma levels.

Substantially reduced risk of cancer of the aerodigestive tract in subjects with variant--463A of the myeloperoxidase gene.

Myeloperoxidase (MPO), an enzyme that is highly expressed in neutrophil leukocytes, transforms precarcinogens such as benzo(a)pyrene and aromatic amines to highly reactive intermediates. A G/A polymorphism located 463 bp upstream of exon 1 in the promoter region strongly reduces MPO mRNA expression. In a matched case-control study, 196 lung cancer, 245 laryngeal cancer, and 255 pharyngeal cancer patients from the Berlin area were investigated for frequency of the G-463A polymorphism by PCR/RFLP, using AciI.

Simultaneous assessment of CYP3A4 and CYP1A2 activity in vivo with alprazolam and caffeine.

Alprazolam (ALP) and caffeine (CAF) were suggested as probe drugs for the activities of CYP3A4 and CYP1A2, respectively. We investigated the disposition of oral ALP (1 mg) and CAF (100 mg) in 17 normal volunteers to establish and validate a procedure for the simultaneous assessment of CYP3A4 and CYP1A2 enzyme activity. Nine received ALP alone, ALP and CAF and CAF alone in an open three-way crossover study to test for pharmacokinetic interaction.

Population frequency, mutation linkage and analytical methodology for the Arg16Gly, Gln27Glu and Thr164Ile polymorphisms in the beta2-adrenergic receptor among Turks.

Aims: Inherited polymorphisms of codons 16, 27, and 164 of the beta2-adrenergic receptor (B2AR) gene may result in significantly changed functions of this receptor. The aim of the present study was to investigate the frequencies of the main mutations of the B2AR gene in Turks.

Methods: A group of 104 unrelated Turkish subjects were analysed for the Arg16Gly, Gln27Glu, and Thr164Leu polymorphisms of B2AR by a newly designed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay.

Omeprazole weakly inhibits CYP1A2 activity in man.

Background And Objectives: Omeprazole is an inducer of human cytochrome P450 1A (CYP1A) enzymes, but shows inhibitory effects on CYP2C19 and CYP3A4. In this study, a potential inhibitory effect of omeprazole on caffeine metabolism, a validated CYP1A2 marker, was examined.

Methods: A randomized, balanced crossover single-dose study was conducted in 16 healthy volunteers comprising 12 extensive (EM) and 4 poor metabolizers (PM) for CYP2C19.

Correlation between genotype and phenotype of the human arylamine N-acetyltransferase type 1 (NAT1).

Arylamine N-acetyltransferase 1 (NAT1) conjugates several aromatic amines and their N-hydroxylated metabolites by N- or O-acetylation. NAT1 genotype and phenotype is known to be variable in human populations. In this study, we set out to measure the functional relevance of the frequent NAT1 gene variants for the activity in human red blood cells.

Flavin monooxygenase 3 (FMO3) polymorphism in a white population: allele frequencies, mutation linkage, and functional effects on clozapine and caffeine metabolism.

Aim: The flavin-containing monooxygenase 3 (FMO3) has been shown to be genetically polymorphic. In vitro, the enzyme contributes to the N-oxidation of clozapine, caffeine, and several other drugs. We therefore wanted to analyze population frequencies and allelic linkage of FMO3 mutations and their functional effect on the metabolism of clozapine and caffeine.

Pharmacokinetic interaction of digoxin with an herbal extract from St John's wort (Hypericum perforatum).

Objective: Extracts of St John's wort (Hypericum perforatum) are widely used in the treatment of depression, often as an over-the-counter drug. In contrast to its frequent use, knowledge about the pharmacokinetics of ingredients and drug interactions of St John's wort is poor. We studied the interaction between hypericum extract LI160 and digoxin.

Frequency of cytochrome P450 CYP2C9 variants in a Turkish population and functional relevance for phenytoin.

Aims: The genetically polymorphic cytochrome P450 enzyme CYP2C9 metabolizes many important drugs. We studied the frequency of the amino acid variants cysteine144 (CYP2C9*2 ) and leucine359 (CYP2C9*3 ) in a Turkish population and the correlation between genotype and phenotype using phenytoin as probe drug.

Methods: CYP2C9 alleles *2 and *3 were measured in 499 unrelated Turkish subjects by PCR and restriction fragment length pattern analysis.

Low frequency of defective alleles of cytochrome P450 enzymes 2C19 and 2D6 in the Turkish population.

Background And Objectives: The genetically polymorphic cytochrome P450 enzymes 2Cl9 (CYP2Cl9) and 2D6 (CYP2D6) contribute to the metabolism of about 30% of all drugs. For analysis of the ethnic-related differences in drug disposition and as a preparation for routine genotyping, we examined CYP2C19 and CYP2D6 mutations in a large Turkish population.

Methods: CYP2C19 and CYP2D6 alleles were determined with use of genomic deoxyribonucleic acid from 404 unrelated Turkish individuals.

Functional significance of a C-->A polymorphism in intron 1 of the cytochrome P450 CYP1A2 gene tested with caffeine.

Aims: The cytochrome P450 enzyme CYP1A2 metabolises several drugs and carcinogens. We wanted to determine how much of the variability of CYP1A2 activity is explained by a newly discovered gene polymorphism in intron 1.

Methods: A single nucleotide polymorphism in intron 1 of the CYP1A2 gene at position 734 downstream of the first transcribed nucleotide was identified by DNA sequence analysis.

Detection of the GSTM1*0 allele by long polymerase chain reaction.

Approximately 50% of individuals in most human populations completely lack activity of the detoxifying enzyme glutathione S-transferase M1. The medical consequences of this deficiency have been extensively investigated in molecular epidemiological studies, but possible differences of the highly active homozygous genotype versus the moderately active heterozygous genotype could not be considered because many currently used polymerase chain reaction (PCR) assays cannot distinguish the homozygous genotypes GSTM1 *A/A and GSTM1*B/B from the heterozygous genotypes GSTM1*A/0 and GSTM1*B/0. Here, we describe that long PCR is suitable for this purpose by specifically producing a signal for the GSTM1*0 allele.

High benzo[a]pyrene diol-epoxide DNA adduct levels in lung and blood cells from individuals with combined CYP1A1 MspI/Msp-GSTM1*0/*0 genotypes.

Levels of anti-benzo[a]pyrene diol-epoxide DNA adducts were analysed by high-pressure liquid chromatography/fluorimetric detection in non-tumorous lung tissues from 20 lung cancer patients and in white blood cells from 20 polycyclic aromatic hydrocarbon exposed coke oven workers. All were current tobacco smokers. CYP1A1 mutations (MspI at 6235 nt, Ile-Val462) and GSTM1 deletion polymorphisms in each individual were analysed in genomic DNA by PCR/restriction fragment length polymorphism.

Polymorphisms in xenobiotic conjugation and disease predisposition.

Low activity of arylamine N-acetyltransferase 2 (slow NAT2) was consistently associated with urinary bladder cancer risk. The increased cancer risk attributable to slow NAT2 was more significant when taking gene-environment interactions and gene-gene interactions into account. In urinary bladder, slow NAT2 was no risk factor in subjects who never smoked but became increasingly relevant with increasing lifetime dose of tobacco smoke expressed by an odds ratio of 2.

Quantification of the antipsychotics flupentixol and haloperidol in human serum by high-performance liquid chromatography with ultraviolet detection.

A high-performance liquid chromatography (HPLC) method was developed for quantification of both isomers of the thioxanthene neuroleptic flupentixol and of the butyrophenone derivative haloperidol in human serum. After extraction with diethyl ether-n-heptane (50:50, v/v), an isocratic normal-phase HPLC system with a Hypersil cyanopropyl silica column (250x4.6 mm, 5 microm particle size) was used with ultraviolet detection at 254 nm and elution with a mixture of 920 ml acetonitrile, 110 ml methanol, 30 ml 0.

Prediction of aryl hydrocarbon receptor-mediated enzyme induction of drugs and chemicals by mRNA quantification.

Enzyme-specific testing for drug interactions by in vitro techniques has become a routine practice in drug development. With many drugs, enzyme induction has similar importance for the prediction of drug-drug interactions. We developed a method for recognizing enzyme induction mediated via the aryl hydrocarbon receptor.

Concordance between enzyme activity and genotype of glutathione S-transferase theta (GSTT1).

Blood samples from 140 healthy German volunteers were used to further characterize the genetic polymorphism of the human theta class glutathione S-transferase 1 (GSTT1). For measurements of GSTT1 activity, hemolysates were incubated in vitro with different concentrations of dichloromethane. The resulting enzymatically mediated production of formaldehyde was determined colorimetrically by the Nash reaction.

Hypericin and pseudohypericin: pharmacokinetics and effects on photosensitivity in humans.

Extracts of St. John's wort (Hypericum perforatum) are used in treatment of depression. They contain various substances with the naphthodianthrones hypericin and pseudohypericin as characteristic ingredients.

CYP1A1 mutations 4887A, 4889G, 5639C and 6235C in the Polish population and their allelic linkage, determined by peptide nucleic acid-mediated PCR clamping.

Mutations in the CYP1A1 gene were investigated in 324 Polish children and adolescents using PCR/RFLP. Mutation T6235C (m1) occurred in 6.6% of alleles (95& confidence limits 4.

Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences.

Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs. CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles. Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine.

Deficiency of glutathione S-transferases T1 and M1 as heritable factors of increased cutaneous UV sensitivity.

Glutathione S-transferases (GSTs) play a primary role in cellular defense against electrophilic chemical species and radical oxygen species. Because free radical attack is one mechanism of UV irradiation-caused skin damage, we investigated whether genetic variation at the GST loci GST T1 and GST M1 influences individual UVB sensitivity. In a double-blind clinical trial, 50 healthy volunteers were evaluated for minimal erythema dose of UVB irradiation, MED (J/cm2), skin types were assigned, and internal standard-controlled polymerase chain reaction (PCR) was used to identify their GST T1 and GST M1 genotypes.