Debrisoquine 4-hydroxylase Research Articles

Associations between Selected ADRB1 and CYP2D6 Gene Polymorphisms in Children with Ventricular and Supraventricular Arrhythmias.

Background and Objectives: Tachycardia is a common cardiovascular disease. Drugs blocking β1-adrenergic receptors (ADRB1) are used in the therapy of arrhythmogenic heart diseases. Disease-related polymorphisms can be observed within the ADRB1 gene. The two most important are Ser49Gly and Arg389Gly, and they influence the treatment efficacy. The family of the cytochrome P450 system consists of the isoenzyme CYP2D6 (Debrisoquine 4-hydroxylase), which is involved in phase I metabolism of almost 25% of clinically important drugs, including antiarrhythmic drugs. A study was conducted to detect the ADRB1 and CYP2D6 gene polymorphisms. Materials and Methods: The material for the test was whole blood from 30 patients with ventricular and supraventricular tachycardia and 20 controls. The samples were obtained from the Department of Pediatric Cardiology. The first to be made was the extraction of DNA using a GeneMATRIX Quick Blood DNA Purification Kit from EURx. The selected ADRB1 and CYP2D6 gene polymorphisms were detected by high-resolution melting polymerase chain reaction (HRM-PCR) analysis. Results: Based on the analysis of melt profile data for each PCR product, the identification of polymorphisms was carried out. Heterozygotes and homozygotes were found in the examined alleles. Conclusions: The frequency of the Arg389Gly polymorphism differs statistically significantly between the control group and patients with supraventricular and ventricular arrhythmias, as well as between these two groups of patients. Moreover, the Arg389Gly polymorphism was statistically more prevalent in the group of girls with SVT arrhythmia compared to girls with VT. A few carriers of homozygous and heterozygous systems of the S49G polymorphism were detected among patients with arrhythmias, as well as control group. The percentage of individuals carrying the CYP2D6 4 allele as either homozygous or heterozygous was observed in the study and control groups. The high prevalence of the CYP2D6*4 allele carriers in both groups prompts the optimization of beta-1 blocker therapy.

Further evidence for the association of CYP2D6*4 gene polymorphism with Parkinson\u2019s disease: a case control study

BackgroundGenetic and environmental risk factors play an important role for the susceptibility to sporadic Parkinson’s disease (PD). It was hypothesized that a splice variant of the CYP2D6 gene (CYP2D6*4 allele) is associated with PD because it alters the ability to metabolize toxins and in particular neurotoxins. CYP2D6 codes for the drug metabolizing enzyme debrisoquine 4-hydroxylase. The CYP2D6*4 variant results in an undetectable enzyme activity and consequently in a reduction in metabolism of some toxins.MethodsSome of agricultural chemicals have neurotoxic potential and CYP2D6 is involved in their detoxification. Thus, we conducted a case control study to investigate the association of the CYP2D6*4 with PD in a Pakistani subpopulation that is known to be exposed to high levels of some agricultural pesticides, insecticides and herbicides.ResultsWe found a significantly higher allele and genotype frequency of the CYP2D6*4 variant in 174 sporadic PD patients when compared to 200 controls. In addition, there was a trend to an earlier age of PD onset and a tremor dominant phenotype in CYP2D6*4 variant carriers.ConclusionOur data provide further evidence that a poor metabolizer status may increase the risk to develop PD especially in populations that are exposed to environmental toxins.

Characterization of the CYP2D6 drug metabolizing phenotypes of the Chilean mestizo population through polymorphism analyses

We tested the influence of four polymorphisms and gene duplication in CYP2D6 on in vivo enzyme activity in a Chilean mestizo population in order to identify the most relevant genetic profiles that account for observed phenotypes in this ethnic group.CYP2D6*2 (2850C>T), *3 (2549A>del), *4 (1846G>A), *17 (1023C>T) and gene duplication were determined by PCR-RFLP or PCRL in a group of 321 healthy volunteers. Individuals with different variant alleles were phenotyped by determining debrisoquine 4-hydroxylase activity as a metabolic ratio (MR) using a validated HPLC assay.Minor allele frequencies were 0.41, 0.01, 0.12 and 0.00 for CYP2D6*2, *3, *4 and *17 variants, respectively, and the duplication frequency was 0.003. Genotype analysis correlated with phenotypes in 18 of 23 subjects (78.3%). 11 subjects were extensive metabolizers (EM), 8 were intermediate metabolizers (IM), 2 were poor metabolizers (PM) and 2 were ultra-rapid metabolizers (UM) which is fairly coincident with expected phenotypes metabolic ratios ranged from 0.11 to 126.41. The influence of CYP2D6*3 was particularly notable, although only heterozygote carriers were present in our population. Individuals homozygous for *4 were always PM. As expected, the only subject with gene duplication was UM.In conclusion, there was a clear effect of genotype on observed CYP2D6 activity. Classification of EM, PM and UM through genotyping was useful to characterize CYP2D6 phenotype in the Chilean mestizo population.

Polymorphic genes of detoxification and mitochondrial enzymes and risk for progressive supranuclear palsy: a case control study

BackgroundThere are no known causes for progressive supranuclear palsy (PSP). The microtubule associated protein tau (MAPT) H1 haplotype is the major genetic factor associated with risk of PSP, with both oxidative stress and mitochondrial dysfunction also implicated. We investigated whether specific single nucleotide polymorphisms (SNPs) in genes encoding enzymes of xenobiotic detoxification, mitochondrial functioning, or oxidative stress response, including debrisoquine 4-hydroxylase, paraoxonase 1 and 2, N-acetyltransferase 1 and 2 (NAT2), superoxide dismutase 1 and 2, and PTEN-induced putative kinase are associated with PSP.MethodsDNA from 553 autopsy-confirmed Caucasian PSP cases (266 females, 279 males; age at onset 68 ± 8 years; age at death 75 ± 8) from the Society for PSP Brain Bank and 425 clinical control samples (197 females, 226 males; age at draw 72 ± 11 years) from healthy volunteers were genotyped using Taqman PCR and the SequenomiPLEX Gold assay.ResultsThe proportion of NAT2 rapid acetylators compared to intermediate and slow acetylators was larger in cases than in controls (OR = 1.82, p < 0.05). There were no allelic or genotypic associations with PSP for any other SNPs tested with the exception of MAPT (p < 0.001).ConclusionsOur results show that NAT2 rapid acetylator phenotype is associated with PSP, suggesting that NAT2 may be responsible for activation of a xenobiotic whose metabolite is neurotoxic. Although our results need to be further confirmed in an independent sample, NAT2 acetylation status should be considered in future genetic and epidemiological studies of PSP.

The association between Parkinson's disease and melanoma

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a loss of melanin-positive, dopaminergic neurons in the substantia nigra. Although there is convincing epidemiologic evidence of a negative association between PD and most cancers, a notable exception to this is that melanoma, a malignant tumor of melanin-producing cells in skin, occurs with higher-than-expected frequency among subjects with PD and that melanoma patients are more likely to have PD. A clear biological explanation for this epidemiological observation is lacking. Here, we present a comprehensive review of published literature exploring the association between PD and melanoma. On the basis of published findings, we conclude that (i) changes in pigmentation including melanin synthesis and/or melanin synthesis enzymes, such as tyrosinase and tyrosine hydroxylase, play important roles in altered vulnerability for both PD and melanoma; (ii) changes of PD-related genes such as Parkin, LRRK2 and α-synuclein may increase the risk of melanoma; (iii) changes in some low-penetrance genes such as cytochrome p450 debrisoquine hydroxylase locus, glutathione S-transferase M1 and vitamin D receptor could increase the risk for both PD and melanoma and (iv) impaired autophagy in both PD and melanoma could also explain the association between PD and melanoma. Future studies are required to address whether altered pigmentation, PD- or melanoma-related gene changes and/or changes in autophagy function induce oncogenesis or apoptosis. From a clinical point of view, early diagnosis of melanoma in PD patients is critical and can be enhanced by periodic dermatological surveillance, including skin biopsies.

Role of the CYP2D6, EPHX1, MPO, and NQO1 genes in the susceptibility to acute lymphoblastic leukemia in Brazilian children

Polymorphic variations of several genes associated with dietary effects and exposure to environmental carcinogens may influence susceptibility to leukemia development. The objective of the present study was to evaluate the effect of the polymorphisms of debrisoquine hydroxylase (CYP2D6), epoxide hydrolase (EPHX1), myeloperoxidase (MPO), and quinone-oxoreductase (NQO1), which have been implicated in xenobiotic metabolism, on the risk of childhood acute lymphoblastic leukemia (ALL). We evaluated the frequency of polymorphisms in the CYP2D6 (*3 and *4), EPHX1 (*2 and *3), MPO (*2), and NQO1 (*2) genes in 206 patients with childhood ALL and in 364 healthy individuals matched for age and gender from a Brazilian population separated by ethnicity (European ancestry and African ancestry), using the PCR-RFLP method. The CYP2D6 polymorphism variants were associated with an increased risk of ALL. The EPHX1, NQO1, and MPO variant genotypes were significantly associated with a reduced risk of childhood ALL. A significantly stronger protective effect is observed when the EPHX1, NQO1, and MPO variant genotypes are combined suggesting that, CYP2D6 polymorphisms may play a role in the susceptibility to pediatric ALL, whereas the EPHX1, NQO1, and MPO polymorphisms might have a protective function against leukemogenesis.

Identification of CYP2D6 null variants among long‐stay, chronic psychiatric inpatients: Is it strictly necessary?

We identified the null variants *3,*4,*5,*6,*7 and *8 of the CYP2D6 gene [encoding for cytochrome P450 (debrisoquine hydroxylase)] in a group of 84 chronic-stay psychiatric inpatients with severe schizophrenia or related disorders and receiving treatment with one or more CYP2D6 substrates for years. We also studied a group of 100 healthy controls of similar ethnic origin (Spanish Caucasians). Three patients were poor metabolizers (PMs) for antipsychotic drugs according to their CYP2D6 genotype (i.e. homozygous for the *4 allele) but they exhibited no adverse drug reaction over the years despite chronic treatment with CYP2D6 substrates. We suggest that CYP2D6 genetic screening is more useful in other type of psychiatric patients, particularly in younger ones starting treatment protocols.

Genotyping of the cytochrome P450 2D6 4469 C>T polymorphism using SimpleProbes™

Background. Genotyping of human cytochrome P450s is a pharmacogenetic approach to diagnosing inherited deficiencies in drug metabolizing enzymes that influence therapeutic responses. The P450 CYP2D6 (debrisoquine hydroxylase) metabolizes numerous antidepressants and neuroleptic agents and there is evidence of a relationship between gene polymorphism and variant therapeutic response. Polymorphism in CYP2D6 causes poor, intermediate, efficient or ultrarapid metabolization of substrate drugs affecting pharmacokinetic parameters and requiring dose adjustments. Predictive genotyping for broader clinical application is reliant on fast, technically simple analyses. A new genotyping method was explored. It identifies the single nucleotide polymorphism (SNP) 4469 C>T (NCBI access no. M33388) with one fluorescent hybridization probe (SimpleProbes™; SP) using the LightCycler™ (LC). This SNP is found in 21 alleles, comprising 30% in Caucasian populations and encoding enzymes with poor, intermediate or efficient activity. The remaining 65 known alleles either harbour a C in position 4469 or are deletion mutants. Methods. Comparative detection of C>T polymorphism was done using a well‐established polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) technique and PCR followed by melting‐point (Tm) analysis with an SP covering the SNP position in 144 samples encompassing alleles *2 and *41 with a T, alleles *1,*3, *4, *6, *9, *10, *15 with a C and the deletion mutant allele *5. Results. C>T polymorphism was detected with complete concordance. Tm of SP/target heteroduplex complexes for C was: Tm 67, 89°C to 68, 62°C and for T: Tm 60, 70°C to 61, 51°C. Conclusion. By one‐step SP methodology it proved possible within 2 h to identify an SNP in genotypes comprising >90% in Caucasian populations.

Association of CYP2D6 100 C &gt; T and 2850 C &gt; T polymorphisms with generalized tonic clonic seizures among Indians

Multiple avenues of research have provided evidence for the role of genetic and environmental factors in epilepsy. Previous studies indicated an association of debrisoquine hydroxylase (CYP2D6) with susceptibility to epilepsy. In this study, association of CYP2D6 100C > T and 2850C > T polymorphisms with generalized tonic clonic seizures (GTCS) among Indians has been analysed using case-control approach. A significant association of 2850C > T (P = 0.015) has been observed. Comparison between phenytoin toxic and others among patients showed no association of these polymorphisms with phenytoin toxicity.

Application of microarray technology in psychotropic drug trials

Microarrays can be manufactured to detect hundreds of thousands of polymorphisms in DNA from patients in psychotropic drug trials. Some of these polymorphisms may be useful as pharmacogenetic predictors of treatment outcomes. We tested a microarray designed to detect common polymorphisms in the CYP2D6 gene that encodes debrisoquine hydroxylase (DH). DH is involved in the hepatic metabolism of many psychotropics. CYP2D6 genotypes predicted plasma steady state concentrations of nortriptyline, a classic DH substrate, in a sample of geriatric patients with major depression. However, in a sample of 246 geriatric patients treated with paroxetine or mirtazapine, both of which are metabolized in part by DH, CYP2D6 genotypes determined with microarrays did not predict discontinuations due to adverse events or severity of adverse events. For modern antidepressants such as paroxetine and mirtazapine, pharmacokinetic factors that are regulated by CYP2D6 such as plasma drug concentrations may be less important than pharmacodynamic factors in determining outcomes. Studies of single candidate genes such as CYP2D6 have only begun to utilize the potential of microarrays for pharmacogenetic prediction. Yet, there is controversy as to whether genome-wide studies designed to detect millions of genotypes with microarrays will lead to new pharmacogenetic discoveries, or whether a more focused, hypothesis-driven approach is better.

Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification

An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM–ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n + 1 primers are used for an n-plex PCR amplification. The specificity of ALM–ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM–ASA. The results for genotyping 70 different genome samples by the 17-plex ALM–ASA were completely consistent with those obtained by both Sanger’s sequencing and PCR restriction fragment length polymorphism (PCR–RFLP) analysis. ALM–ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM–ASA with a well-developed automation device for sample processing.

Parkinson's disease risk factors: genetic, environmental, or both?

Perhaps one of the most important questions posed by the neurobiology of aging concerns the pathogenic mechanisms in Parkinson's disease (PD). Recently, it was suggested that exposure to pesticides could be the main cause of PD. Another study reported that the environmental endotoxin, lipopolysaccaride produced by Salmonella minnesota, might be a risk factor for PD. An alternative explanation is the genetic component, which has been suggested to be an important risk factor. Epidemiological studies have identified a positive family history of Parkinson as one of the most important risk factors for the disease. However, these studies neither examined nor reviewed the medical records of the family members. The twin study stated that the major factors in the etiology of PD are non-genetic. Meanwhile, epidemiological studies from China have shown that the prevalence of PD is much lower than in the Caucasian population, explained by the low frequency of cytochrome P-450 CYP2D6 debrisoquine hydroxylase gene polymorphism. The etiology of idiopathic PD is still a question for scientists, and calls for further research, especially with the growing proportion of elderly and the rising incidence of PD worldwide. Future research for PD risk factors should consider that multiple interactions occur in PD, resulting in a complex trait, which includes genetic, acquired, and environmental components.

Influence of phenylalanine 120 on cytochrome P450 2D6 catalytic selectivity and regiospecificity: crucial role in 7-methoxy-4-(aminomethyl)-coumarin metabolism

The polymorphic human debrisoquine hydroxylase, cytochrome P450 2D6 (CYP2D6), is one of the most important phase I drug metabolising enzymes. It is responsible for metabolising a large number of compounds that mostly share similarity in having a basic N-atom and an aromatic moiety. In homology modelling studies, it has been suggested that in fixation of this aromatic moiety, there may be an important role for phenylalanine 120 (Phe 120). In this study, the role of Phe 120 in ligand binding and catalysis was experimentally examined by mutating it into an alanine. Strikingly, this substitution led to a completely abolished 7-methoxy-4-(aminomethyl)-coumarin (MAMC) O-demethylating activity of CYP2D6. On the other hand, bufuralol metabolism was hardly affected ( K m of 1-hydroxylation mutant: 1.2 μM, wild-type: 2.9 μM, 4-hydroxylation mutant: 1.5 μM, and wild-type: 3.2 μM) and neither was affected dextromethorphan O-demethylation ( K m mutant: 1.2 μM, wild-type: 2 μM, k cat mutant: 4.5 min −1, and wild-type: 3.3 min −1). However, the Phe 120Ala mutant also formed 3-hydroxymorphinan, the double demethylated form of dextromethorphan, which was not detected using wild-type CYP2D6. 3,4-Methylenedioxymethamphetamine (MDMA) was demethylenated by both mutant and wild-type CYP2D6 to 3,4-dihydroxymethamphetamine (3,4-OH-MA K m of mutant: 55 μM and wild-type: 2 μM). In addition, the mutant formed two additional metabolites; 3,4-methylenedioxyamphetamine (MDA) and N-hydroxy-3,4-methylenedioxymethamphetamine ( N-OH-MDMA). Inhibition experiments of dextromethorphan O-demethylation showed a decreased affinity of the Phe 120Ala mutant for quinidine (IC 50 mutant: 240 nM and wild-type, 40 nM), while IC 50s for quinine were equal (1 μM). These data indicate the importance of Phe 120 in the selectivity and regiospecificity in substrate binding and catalysis by CYP2D6.

Fatal intoxication cases: cytochrome P450 2D6 and 2C19 genotype distributions.

Many commonly used pharmaceuticals, such as antidepressants and neuroleptics as well as some illegal drugs, are metabolised by the cytochrome P450 enzyme debrisoquine 4-hydroxylase (CYP2D6). Of Caucasians, 7-10% lack this enzyme, which can, upon administration of drugs in normal therapeutic doses, lead to adverse reactions and unexpected intoxication, leading in turn even to a fatal outcome in some cases. Individuals (n=242) who had died due to intoxication by pharmaceuticals were genotyped for CYP2D6 and CYP2C19 and compared with a reference group of 281 blood donors. A single nucleotide polymorphism (SNP) method was used to identify five CYP2D6 alleles: *1 (wt), *2, *3, *4 and *6. The allele *5, a complete gene deletion, was identified by a multiplex amplification of long DNA fragments. Four CYP2C19 alleles *1 (wt), *2, *3 and *4 were also identified by SNP analysis. The prevalence of the CYP2D6 poor metaboliser (PM) genotypes in individuals with fatal intoxication was lower (4.7%) than expected from the frequencies of these genotypes in the blood donors (8.5%). A significantly lower frequency P<0.005 (0.03 with correction according to Bonferroni) was found for the CYP2D6*4 allele among the fatal intoxication cases. The CYP2C19 genotype analyses showed the same results for the fatal intoxication cases and for the blood donors. The findings in this study confirm our earlier observations of a lower frequency of CYP2D6 PM genotypes in cases of fatal intoxication. To our knowledge, it has not been shown previously that intoxication victims might have a lower frequency of PMs than the general population.

Cloning and functional expression of a novel marmoset cytochrome P450 2D enzyme, CYP2D30: comparison with the known marmoset CYP2D19

Using a primer set designed on the cDNA encoding the known marmoset cytochrome P450 2D19 (CYP2D19), a cDNA encoding a novel CYP2D enzyme (CYP2D30) was cloned from the liver of a female marmoset bred at Kyoto University (KYU). In addition, a cDNA encoding CYP2D19 was cloned from the liver of a female marmoset bred at Kagoshima University (KAU). CYP2D30 and CYP2D19 showed homologies of 93.6 and 93.4% in their nucleotide and amino acid sequences, respectively. Reverse transcription polymerase chain reaction (RT-PCR) and digestion with NdeI demonstrated that the KYU-marmoset liver contained mainly mRNA for CYP2D30, while the KAU-marmoset liver contained mainly mRNA for CYP2D19. Marmoset CYP2D30, like human CYP2D6, exhibited high debrisoquine (DB) 4-hydroxylase activity and relatively low DB 5-, 6-, 7- and 8-hydroxylase activities, whereas CYP2D19 lacked DB 4-hydroxylase but exhibited marked 5-, 6-, 7- and 8-hydroxylase activities. The two marmoset recombinant enzymes showed enantioselective bufuralol (BF) 1″-hydroxylase activities, similar to CYP2D6. BF 1″-hydroxylation by CYP2D30 exhibited product-enantioselectivity of (1″ R-OH-BF ⪡ 1″ S-OH-BF), similar to that observed with human CYP2D6, whereas CYP2D19 showed a reversed selectivity of (1″ R-OH-BF ≥ 1″ S-OH-BF). BF 1″-hydroxylation in marmoset liver microsomes from both sources was inhibited by antibodies raised against rat CYP2D1 in a concentration-dependent manner. A known inhibitor of CYP2D6, quinidine, effectively inhibited the BF 1″-hydroxylation activities in liver microsomal fractions prepared from KYU- and KAU-marmosets. These results suggest that CYP2D19 and CYP2D30 proteins can be expressed as functional enzymes in marmoset livers, although it is unresolved whether both enzymes coexist in the same marmoset liver.

Relationship between Type A and B personality and debrisoquine hydroxylation capacity

A person with Type A personality is an 'aggressor' compared with the rarely harried Type B. Although debrisoquine hydroxylase (CYP2D6) capacity has been associated with personality, no study has specifically investigated its association with personality Type A and B. Therefore the aim of this research was to study the impact of CYP2D6 on Type A and B personality. Type A and B personality questionnaires were administered to 48 healthy patients undergoing elective orthopaedic surgery. After obtaining informed consent, patients were genotyped for the various CYP2D6 alleles by allele-specific polymerase chain reaction. Based on the genotypes, patients were grouped as extensive metabolizer (EM)1 (normal) (CYP2D6*1/*1), EM2 (intermediate) (CYP2D6*1/*4, CYP2D6*1/*5, CYP2D6*1/*9 and CYP2D6*1/*10) and EM3 (slow) (CYP2D6*4/*10, CYP2D6*5/*10, CYP2D6*10/*10 and CYP2D6*10/*17). Chi(2) was used to determine the relationship between the groups and personality types. The percentages of patients who were of the EM1, EM2 and EM3 groups were 20.8%, 52.1% and 27.1%, respectively. There was a significant difference (P = 0.032) between the three groups in terms of personality type, in which EM1 showed a tendency to be of personality Type A while EM2 and EM3 tended to be of personality Type B. The study suggests that there is a relationship between CYP2D6 activity and Type A and B personality.

Cytochrome P450 2D6 genotyping of fatal intoxications using Pyrosequencing

Many commonly used pharmaceuticals such as antidepressants and neuroleptics as well as some illegal drugs are metabolised by the Cytochrome P450 enzyme debrisoquine 4-hydroxylase (CYP2D6). Seven to ten percent of Caucasians lack this enzyme which can lead to adverse reactions and in some cases to unexpected intoxication even with fatal outcome, upon administration of drugs in normal therapeutic doses. 236 individuals who had died due to intoxication of pharmaceuticals were genotyped for CYP2D6 and compared to a reference group of 281 blood donors. A single nucleotide polymorphism (SNP) method was used to identify five CYP2D6 alleles: *1 (wt), *2, *3, *4 and *6. The allele *5, a complete gene deletion, was identified by a multiplex amplification of long DNA fragments. The prevalence of the CYP2D6 PM genotype in the fatal intoxications was lower (4.7%) compared to the blood donors (8.5%). A significant decrease ( p<0.005) was found in the CYP2D6*4 allele frequency among the fatal intoxications.

Changes in the Enzymatic Properties of CYP2D6 by the Substitution of Phenylalanine at Position 120 by Alanine

The functional roles of phenylalanine at position 120 (Phe-120) in the oxidation of bunitrolol (BTL), debrisoquine (DB) and bufuralol (BF) by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system (Saccharomyces cerevisiae AH-22 strain). The substitution of Phe-120 by alanine markedly increased the activities of enantiomeric BTL 4-hydroxylase and DB 4-hydroxylase, whereas it did not remarkably affect BF 1′′-hydroxylase activities. Kinetic studies revealed that the substitution of Phe-120 by alanine increased the Km and Vmax values for enantiomeric BTL 4-hydroxylation, but increased only the Vmax value for DB 4-hydroxylation without changing the Km value. Km and Vmax values for BF 1′′-hydroxylation were similar between the mutant and the wild-type. The dissociation constants of the mutant calculated from the binding spectra for BTL enantiomers were higher than those of the wild-type, suggesting that the substitution of Phe-120 by alanine decreased the affinity of CYP2D6 for BTL enantiomers. These results indicate that Phe-120 has an important role in the oxidation of substrates by CYP2D6.

Cytochrome P450 expression and testosterone metabolism in the liver of deer

Cytochrome P450 expression in cervine liver was investigated using chemical probes and Western blot analysis, and compared with the rat. Deer liver, when compared with rat liver, was characterised by high ethoxyresorufin O-deethylase, coumarin 7-hydroxylase and, to a lesser extent, erythromycin N-demethylase activities; in contrast, deer liver exhibited low debrisoquine 4-hydroxylase, chlorzoxazone 6-hydroxylase and, particularly, lauric acid hydroxylase activities. Ethoxyresorufin O-deethylase activity in deer was markedly inhibited by α-naphthoflavone, but was relatively resistant to inhibition by furafylline. Coumarin 7-hydroxylase was inhibited by 8-methoxypsoralen. Western blot analysis using antibodies to rat CYP1A recognised a single, highly expressed protein. Kinetic analysis indicated that a single enzyme is likely to be responsible for the high ethoxyresorufin O-deethylase activity in deer liver. Probing of cervine hepatic microsomes with antibodies to rat CYP2A2 showed that apoprotein levels were higher in the deer compared with the rat. Eadie–Hofstee plot analysis indicated that more than one enzyme catalyses the 7-hydroxylation of coumarin. Western blot analysis using antibodies to rat CYP2B, rat CYP2C11, human CYP2D6, rat CYP3A and rat CYP4A1 revealed in each case the presence of single, poorly expressed, proteins in deer liver. In contrast, when antibodies to rat CYP2E1 were used, a highly expressed single protein was observed. Cervine hepatic microsomes metabolised testosterone to generate androstenedione and a number of hydroxylated products, the major hydroxylation sites being the 2β-, 6β- and possibly the 12-position. In summary, this is the first study showing that deer liver expresses all xenobiotic-metabolising cytochrome P450 families, but the level of expression differs from that of the rat.

No allelic association between harm avoidance and the debrisoquine 4-hydroxylase gene.

Several reports suggest that variance in personality traits is inherited, but little is known about the genes responsible. It has been suggested that there is a relationship between personality characteristics and the gene responsible for the activity of the enzyme debrisoquine 4-hydroxylase (CYP2D6). To examine the proportion of poor metabolizers in a group of personality disordered patients. Blood samples were obtained from 23 patients with a high or very high score on the dimension 'harm avoidance' of the Temperament and Character Inventory (TCI). The samples were genotyped for the null alleles CYP2D6*3 and*4 by performing an allele-specific polymerase chain reaction. The frequencies of genotypes in the sample were very similar to the frequencies found in a general white population. The investigation produced no support for the idea that the CYP2D6 gene is related to personality by means of variations in the temperament dimension of harm avoidance.