Biomedical subjects

A Gaedigk

Publications and source records attributed to A Gaedigk.

31 records · Page 2Linked to original sources

PubMed1998

Comparison of three CYP2D6 probe substrates and genotype in Ghanaians, Chinese and Caucasians.

The ability to metabolize CYP2D6 substrates sparteine, debrisoquine, and dextromethorphan was studied in healthy Caucasian (n = 20), Ghanaian (n = 21), and Chinese (n = 22) CYP2D6 extensive metabolizers. Genotype analysis for the CYP2D6*1, *3, *4, *5, *9, *10, and *17 alleles was performed. Interethnic differences in the disposition of the probe drugs were found among the extensive metabolizers; extensive metabolizer status was confirmed by phenotype and genotype analysis. The mean metabolic rate was lower for Caucasians than for Ghanaians for sparteine (P < 0.02) and for both Ghanaians and Chinese for debrisoquine (P < 0.02). Correlation comparisons resulted in lower pairwise correlation coefficients in Ghanaians compared with Chinese and Caucasians for every combination of probe substrates. In addition, in Chinese and Caucasians, metabolic rates for each pair of probe drugs were significantly correlated (P < 0.002), but in Ghanaians the dextromethorphan metabolic rates were not correlated to either sparteine or debrisoquine (P < 0.05). Even when only those with a CYP2D6*1/*1 genotype were included in the correlation calculations, the Ghanaians had very low correlation coefficients (r(s) - 0.02-0.2, n = 9); much lower than those found in Caucasian (r(s) 0.78-0.92, n = 14) or Chinese (r(s) 0.54-0.96, n = 7) individuals. Quinidine had significantly less affect on sparteine metabolic rates in Ghanaians than both Caucasians and Chinese (P < 0.02). In addition, five of the 21 Ghanaian individuals had dextromethorphan metabolic ratios which were unaffected by quinidine. These individuals also had differences in urinary recovery of dextromethorphan and its metabolites when compared to the other Ghanaian individuals. These results confirm the large ethnic differences in probe drug metabolism and quinidine sensitivity among these ethnic groups. They also suggest that the Ghanaians have an additional unidentified allele(s) with altered substrate specificity and quinidine sensitivity which is currently genotyped as CYP2D6*1.

Adult↗

PubMed1998

Quantification of intraindividual variability and the influence of menstrual cycle phase on CYP2D6 activity as measured by dextromethorphan phenotyping.

Intraindividual variability and the effects of menstrual cycle phase on CYP2D6 activity were evaluated by dextromethorphan phenotyping in 20 Caucasian normal volunteers. Dextromethorphan 30 mg was administered to 10 men every 14 days for 3 months, and to 10 premenopausal women during the mid-follicular and mid-luteal phases of each menstrual cycle for three complete cycles. Urinary dextromethorphan/dextrorphan molar ratios were obtained after an overnight urine collection. Ten women and nine men were extensive metabolizer phenotypes, and one man was a poor metabolizer phenotype (confirmed by genotyping). There was no difference in dextromethorphan metabolic ratios between the mid-follicular (mean +/- SD: 0.00728+/-0.00717) and mid-luteal (0.00745+/-0.00815) phases of the menstrual cycle (P = 0.88). Also, no significant difference was found in the intraindividual variability of the metabolic ratios between the two phases (P = 0.80). No statistically significant sex difference in CYP2D6 activity was found between men (0.00537+/-0.00431) and women (0.00737+/-0.00983) extensive metabolizers (P = 0.84). For all individuals, intraindividual variability in dextromethorphan ratios ranged from 12.1-136.6% with a median of 36.7%. Because hormonal fluctuations within the mid-follicular and mid-luteal phases of the menstrual cycle do not appear to affect CYP2D6 activity, pharmacokinetic or clinical investigations of CYP2D6 substrate activity may not require menstrual cycle phase stratification. Because baseline metabolic ratios may fluctuate an average of 37%, repeat baseline and treatment phenotyping assessments should be obtained for accurate determination of a given drug's effect on CYP2D6 activity when measured by dextromethorphan.

Adult↗

PubMed1998

High frequency of CYP2D6 poor and "intermediate" metabolizers in black populations: a review and preliminary data.

There is little and conflicting information concerning polymorphism of CYP2D6 in populations of Africans and African descent. Estimations of the prevalence of poor metabolizers (PMs) in Black populations have ranged from 0 to 19 percent, and unlike Caucasian and Asian populations, there seems to be a poor correlation in metabolic ratios (MRs) between commonly used CYP2D6 probe drugs. A novel mutant allele, CYP2D6*17, which is associated with reduced metabolic rates, has been determined to occur in high frequencies in African and African American populations. In the present pilot study, there was a high frequency of CYP2D6*17, and about one-third of the African-American participants showed a reduced capacity to metabolize dextromethorphan, a CYP2D6 probe drug. The CYP2D6*17 allele and other variants may possibly play a role in the inconsistent variation of phenotypes in Black populations.

Black People↗

PubMed1997

Human acetyltransferase polymorphisms.

Conjugation of primary amino and hydroxylamino groups with acetate, catalyzed by acetyl CoA-dependent arylamine acetyltransferase (NAT) enzymes, may play an important role in the intricate series of metabolic pathways that produce or prevent toxicity following exposure to homo- and heterocyclic arylamine and hydrazine xenobiotics. Two independently regulated and kinetically distinct human acetyltransferases are now known to exist, namely NAT1 and NAT2. Interindividual variation in NAT2 function is associated with the classical isoniazid acetylation polymorphism which was discovered over forty years ago. At last count, fifteen variant alleles at the NAT2 gene locus have been linked to the isoniazid 'acetylator phenotype', and each of these can be identified in population studies using specific PCR-based genotyping tests. On the other hand, NAT1 shows kinetic selectivity for compounds whose disposition is unrelated to the classical isoniazid acetylation polymorphism. NAT1 expression is also phenotypically variable in human populations, at least in part due to allelic differences at the NAT1 gene locus. Nine NAT1 variant alleles have been described to date, of which NAT1* 14 and NAT1* 15 clearly produce defective NAT1 proteins and lead to functional impairment in the metabolism of NAT1-selective substrates both in vivo and in vitro. On the other hand, it has been reported that the NAT1* 10 variant associates with elevated NAT1 activity and increased risk for cancers of the bladder and colon. Because of the important toxicologic consequences of allelic variation in NAT1 and NAT2 function for the metabolic activation of arylamine and heterocyclic amine procarcinogens, further studies are needed to improve our understanding of the extent of NAT allelic variation, to determine the functional capacity of each variant gene product, and to develop accurate methods of detecting them in population and epidemiological studies.

Acetylation↗

PubMed1997

The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: functional and localization studies.

In both animal models and humans, the first and obligatory step in the activation of arylamines is N-hydroxylation. This pathway is primarily mediated by the phase-I enzymes CYP1A1, CYP1A2 and CYP4B1. In the presence of flavonoids such as alpha-naphthoflavone and flavone, both CYP3A4 and CYP3A5 have also been shown to play a minor role in the activation of food-derived heterocyclic amines. The further activation of N-hydroxyarylamines by phase-II metabolism can involve both N, O-acetylation and N, O-sulfonation catalyzed by N-acetyltransferases (NAT1 and NAT2) and sulfotransferases, respectively. Using an array of techniques, we have been unable to detect constitutive CYP1A expression in any segments of the human gastrointestinal tract. This is in contrast to the rabbit where CYP1A1 protein was readily detectable on immunoblots in microsomes prepared from the small intestine. In humans, CYP3A3/3A4 expression was detectable in the esophagus and all segments of the small intestine. Northern blot analysis of eleven human colons showed considerable heterogeneity in CYP3A mRNA between individuals, with the presence of two mRNA species in some subjects. Employing the technique of hybridization histochemistry (also known as in situ hybridization), CYP4B1 expression was observed in some human colons but not in the liver or the small intestine. Hybridization histochemistry studies have also demonstrated variable NAT1 and NAT2 expression in the human gastrointestinal tract. NAT1 and NAT2 mRNA expression was detected in the human liver, small intestine, colon, esophagus, bladder, ureter, stomach and lung. Using a general aryl sulfotransferase riboprobe (HAST1), we have demonstrated marked sulfotransferase expression in the human colon, small intestine, lung, stomach and liver. These studies demonstrate that considerable variability exists in the expression of enzymes involved in the activation of aromatic amines in human tissues. The significance of these results in relation to a role for heterocyclic amines in colon cancer is discussed.

Amines↗

PubMed1997

Cloning, structural organization, and chromosomal mapping of the human phenol sulfotransferase STP2 gene.

Phenol- and monoamine-metabolizing sulfotransferases (STP and STM, respectively) are members of a superfamily of enzymes that add sulfate to a variety of xenobiotics and endobiotics containing hydroxyl or amino functional groups. To characterize related sulfotransferase genes further, we used extra-long PCR (XL-PCR) to generate three distinct sizes of amplification products from human genomic DNA or from genomic phage library clones, each of which contained sulfotransferase gene sequences. One of the PCR fragments contained a new sulfotransferase gene, STP2, corresponding to a recently published cDNA clone that encodes a sulfotransferase with catalytic specificity distinct from that of the previously described STP1 and STM. Additional upstream sequence information was obtained using a second STP2-specific XL-PCR-based approach. The STP2 gene is composed of eight exons and seven introns, with exon sizes ranging from 95 to 181 bp. Protein-coding exon lengths and locations of the splice junctions were identical to those in both the STM gene and an STP2 gene published independently by another group recently. The STP2 gene maps to a chromosomal location (16p11.2-p12) that is the same as that previously determined for both STP1 and STM. The characterization of the STP2 gene provides further insight into the organization, regulation, and multiplicity of the sulfotransferase supergene family.

Arylsulfotransferase↗

PubMed1997

Tissue-specific expression and alternative splicing of human microsomal epoxide hydrolase.

Human microsomal epoxide hydrolase (HYL1) plays an important role in the detoxification of environmental compounds and drugs, such as the aromatic anticonvulsants phenytoin, carbamazepine, and phenobarbital, by converting their P450-generated epoxide metabolites into nontoxic diols. Recently, we have shown that a genetic defect altering the structure and function of the HYL1 protein is unlikely to be responsible for predisposing individuals to idiosyncratic hypersensitivity reactions from anticonvulsants. To evaluate the possible involvement of regulatory mechanisms, we used 5' rapid amplification of cDNA ends (RACE) and reverse transcription polymerase chain reaction (RT-PCR) to identify and characterize HYL1 5' cDNA ends. In addition to exon 1 (E1) previously isolated from a liver cDNA library, we isolated four new exons (E1-a, E1-c, E1-d, and E1-e) from various tissues. E1 was always directly connected to exon 2 (E2) where the translation start codon is located. E1-a, E1-c E1-d, and E1-e are alternatively spliced to E2, having either E1-a or E1-a' (a truncated form of E1-a) at the 5' end of their respective transcript. Genomic data indicate that exons E1-a and E1-c are located at least 7 kb upstream from E1. Furthermore, we demonstrated a tissue-specific expression pattern for E1-containing mRNA species, whereas E1-a-containing transcripts appear to be expressed ubiquitously. Our results provide evidence that microsomal epoxide hydrolase is regulated by multiple untranslated exons flanked by tissue-specific promoters.

Alternative Splicing↗

PubMed1996

Epitope mapping studies with human anti-cytochrome P450 3A antibodies.

A subset of patients with hypersensitivity reactions to the aromatic anticonvulsants phenytoin, carbamazepine, and phenobarbital have circulating antibodies that recognize members of the rat cytochrome P450 (CYP) 3A subfamily. These antibodies do not recognize related human CYP3A proteins despite the high degree of structural similarity. To investigate the relationship between P450-mediated drug metabolism and the development of anti-P450 antibodies, we initiated epitope mapping studies by screening a library of fusion proteins constructed from rat CYP3A1 with an anti-CYP3A1-positive patient serum sample. Positive signals from colony lifts were confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblotting, and a 26-amino acid sequence corresponding to amino acids 342-367 of the CYP3A1 protein (NKAPPTY-DTVMEMEYLDMVLNETLRL) was identified as containing the epitope recognized by IgG3 antibodies in this serum sample. By subjecting inserts from two clones into a second round of library construction and screening by immunoblot analysis, we further defined the epitope to EYLDMVLNETLRL. Single amino acid deletions identified DMVLNETLRL as the minimum amino acid sequence required for antibody binding. The corresponding sequence in the four human CYP3A proteins differs by only one amino acid (DMVVNETLRL) This amino acid is critical to antibody recognition as immunoreactivity of the L361V mutant is markedly reduced. Anti-CYP3A antibodies in nine of nine additional sera also recognized the 13-amino acid epitope; for five of these sera, the minimum antibody binding sequence was DMVLNETLRL. The proximity of this epitope to a region determining substrate specificity may provide the link among reactive metabolite production, hapten formation, and the production of anti-P450 antibodies in anticonvulsant-induced idiosyncratic reactions.

Amino Acid Sequence↗

PubMed1995

Genetic analysis of microsomal epoxide hydrolase in patients with carbamazepine hypersensitivity.

Carbamazepine therapy is occasionally complicated by hypersensitivity reactions, the mechanism of which is poorly understood. It has been suggested that affected individuals may have a genetically-determined defect of microsomal epoxide hydrolase. The aim of this study was to determine whether a single genetic mutation or pattern of mutations could be used to predict individual susceptibility to carbamazepine-hypersensitivity. DNA was isolated from 10 carbamazepine-hypersensitive patients and 10 healthy volunteers. The patients had developed various forms of toxicity with carbamazepine, including toxic epidermal necrolysis, Stevens-Johnson syndrome, hepatitis and pneumonitis. The technique of polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) was used to screen for mutations in all nine exons of the microsomal epoxide hydrolase gene. Any new mutations detected by this method were characterised by direct sequencing of the DNA. In addition, in the most severely affected patient, we sequenced all nine exons of the gene. There was a higher frequency of mutations in the hypersensitive group when compared with the controls, but there was no consistent mutation (or pattern of mutations) in the microsomal epoxide hydrolase gene which was common to the hypersensitive group. DNA sequencing of all nine exons of the microsomal epoxide hydrolase gene from the most severely affected patient showed the sequence to be "wild-type," when compared to the previously published sequences. The results of this study suggest that a single mutation within the coding region of the microsomal epoxide hydrolase gene cannot be the sole determinant of the predisposition to carbamazepine hypersensitivity.

Adolescent↗

PubMed1994

Characterization of the microsomal epoxide hydrolase gene in patients with anticonvulsant adverse drug reactions.

Therapy with the aromatic anticonvulsants phenytoin, phenobarbital and carbamazepine has been associated with the occurrence of rare idiosyncratic hypersensitivity reactions. These drugs are thought to be activated to potentially reactive arene oxide (epoxide) metabolites by cytochrome P450-dependent monooxygenation, while liver microsomal epoxide hydrolase (mEH) plays a detoxifying role by converting such reactive intermediates to non-toxic dihydrodiols. Evidence from in vitro lymphocyte toxicity tests and enzyme inhibitor studies has suggested that an inherited defect in mEH function may be responsible for the enhanced drug toxicity observed in affected individuals. To test this hypothesis we designed methods to directly compare mEH gene structure in patients presenting with anticonvulsant adverse reactions and in control subjects in which no in vivo or in vitro toxicity to anticonvulsants could be demonstrated. Southern analysis of peripheral lymphocyte DNA using a full-length mEH cDNA as hybridization probe revealed no gross differences in mEH gene structure in selected patients when compared with DNA samples from unaffected control subjects. The human mEH gene was then cloned and characterized from a control individual. Nine exons were identified within a 22 kb region and sequences of selected regions, including all exons, were determined. Single strand conformation polymorphism (SSCP) analysis was performed on all exonic regions in genomic DNA from each of 26 subjects, including six unrelated patients with previous toxicity to anticonvulsant therapy and seven siblings (three of whom had displayed toxicity). Several distinct SSCP patterns could be observed among the subjects tested, each corresponding to a specific point mutation within one of the amplified fragments of the mEH gene. However, none of the SSCP patterns reflecting point mutations was correlated with the occurrence of anticonvulsant toxicity. From these observations we conclude that a genetic defect altering the structure and function of the mEH protein is unlikely to be responsible for predisposing patients to anticonvulsant adverse reactions.

Adult↗

PubMed1991

Debrisoquine/sparteine hydroxylation genotype and phenotype: analysis of common mutations and alleles of CYP2D6 in a European population.

Four different mutations of the cytochrome P450 CYP2D6 gene associated with the poor metabolizer phenotype (PM) of the debrisoquine/sparteine polymorphism were analyzed by Xba I restriction fragment length polymorphism (RFLP) analysis and a polymerase chain reaction (PCR)-based DNA amplification method in DNA of 394 healthy European subjects; 341 of these were phenotyped by sparteine or debrisoquine administration and urinary metabolic ratios (MR). Our study demonstrates the efficiency of the PCR-test for phenotype prediction; 96.4% of individuals were correctly predicted, i.e., 100% of the extensive metabolizers (EMs) and 86.0% of the poor metabolizers (PMs). In contrast, Xba I RFLP analysis was far less informative, predicting the phenotype in only 26.8% of PMs. By combining both DNA tests, the prediction rate of the PM phenotype increased to 90.6%. A point mutation at a splice-site consensus sequence termed D6-B represented the most common mutant CYP2D6 gene and accounted for more than 75% of mutant alleles. In addition, other known mutations such as D6-D (14%), D6-A (5%), and the rare D6-C mutation bring the identified mutant alleles to greater than 95% of all mutant PM-alleles. Most of Xba I 44-kb alleles were confirmed as mutant alleles carrying the D6-B mutation. However, 9.7% did not have this mutation and may express a functional CYP2D6 gene. Moreover, all Xba I 16 + 9-kb alleles contained the D6-B mutation. Heterozygous EM individuals had a significantly higher MR when compared to homozygous EMs. Genotyping provides an important advantage for investigations of the influence of CYP2D6 activity on drug therapy and its association with certain diseases.

Alleles↗

PubMed1991

Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism.

The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs.

Base Sequence↗

PubMed1990

Identification of the primary gene defect at the cytochrome P450 CYP2D locus.

The mammalian cytochrome P450-dependent monooxygenase system is involved in the metabolism of drugs and chemical carcinogens. The role of these enzymes in toxicological response is exemplified by an autosomal recessive polymorphism at the cytochrome P450 CYP2D6 debrisoquine hydroxylase locus which results in the severely compromised metabolism of at least 25 drugs, and which in some cases can lead to life-threatening side-effects. In addition, this polymorphism, which affects 8-10% of the caucasian population, has been associated with altered susceptibility to lung and bladder cancer. Here we report the identification of the primary mutation responsible for this metabolic defect and the development of a simple DNA-based genetic assay to allow both the identification of most individuals at risk of drug side-effects and clarification of the conflicting reports on the association of this polymorphism with cancer susceptibility.

Base Sequence↗