RESUMO
Pharmacogenomics (PGx) investigates the influence of genetics on drug responses, enabling tailored treatments for personalized healthcare. This study assessed the accuracy of genotyping six genes using whole genome sequencing with four different computational tools and various sequencing depths. The effects of using different reference genomes (GRCh38 and GRCh37) and sequence aligners (BWA-MEM and Bowtie2) were also explored. The results showed generally minor variations in tool performance across most genes; however, more notable discrepancies were observed in the analysis of the complex CYP2D6 gene. Cyrius, a CYP2D6-specific tool, demonstrated the most robust performance, achieving the highest concordance rates for CYP2D6 in all instances, comparable to the consensus approach in most cases. There were rather small differences between the samples with 20× coverage depth and those with higher depth, but the decreased performance was more evident at lower depths, particularly at 5×. Additionally, variations in CYP2D6 results were observed when samples were aligned to different reference genomes using the same method, or to the same genome using different aligners, which led to reporting incorrect rare star alleles in several cases. These findings inform the selection of optimal PGx tools and methodologies as well as suggest that employing a consensus approach with two or more tools might be preferable for certain genes and tool combinations, especially at lower sequencing depths, to ensure accurate results. Additionally, we show how the upstream alignment can affect the performance of tools, an important factor to take into account.
Assuntos
Benchmarking , Citocromo P-450 CYP2D6 , Farmacogenética , Humanos , Citocromo P-450 CYP2D6/genética , Farmacogenética/normas , Farmacogenética/métodos , Citocromo P-450 CYP2C19/genética , Sequenciamento Completo do Genoma/normas , Sequenciamento Completo do Genoma/métodos , Técnicas de Genotipagem/métodos , Genótipo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2A6/genética , Testes Farmacogenômicos/normas , Testes Farmacogenômicos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Vitamina K Epóxido RedutasesRESUMO
Tobacco smoking has been highlighted as a major health challenge in modern societies. Despite not causing death directly, smoking has been associated with several health issues, such as cardiovascular diseases, respiratory disorders, and several cancer types. Moreover, exposure to nicotine during pregnancy has been associated with adverse neurological disorders in babies. Nicotine Replacement Therapy (NRT) is the most common strategy employed for smoking cessation, but despite its widespread use, NRT presents with low success and adherence rates. This is attributed partially to the rate of nicotine metabolism by cytochrome P450 2A6 (CYP2A6) in each individual. Nicotine addiction is correlated with the high rate of its metabolism, and thus, novel strategies need to be implemented in NRT protocols. Naturally derived products are a cost-efficient and rich source for potential inhibitors, with the main advantages being their abundance and ease of isolation. This systematic review aims to summarize the natural products that have been identified as CYP2A6 inhibitors, validated through in vitro and/or in vivo assays, and could be implemented as nicotine metabolism inhibitors. The scope is to present the different compounds and highlight their possible implementation in NRT strategies. Additionally, this information would provide valuable insight regarding CYP2A6 inhibitors, that can be utilized in drug development via the use of in silico methodologies and machine-learning models to identify new potential lead compounds for optimization and implementation in NRT regimes.
Assuntos
Citocromo P-450 CYP2A6 , Nicotina , Animais , Humanos , Produtos Biológicos/farmacologia , Citocromo P-450 CYP2A6/antagonistas & inibidores , Citocromo P-450 CYP2A6/metabolismo , Nicotina/metabolismoRESUMO
Dietary exposure to aflatoxin B1 (AFB1) is harmful to the health and performance of domestic animals. The hepatic cytochrome P450s (CYPs), CYP1A1 and CYP2A6, are the primary enzymes responsible for the bioactivation of AFB1 to the highly toxic exo-AFB1-8,9-epoxide (AFBO) in chicks. However, the transcriptional regulation mechanism of these CYP genes in the liver of chicks in AFB1 metabolism remains unknown. Dual-luciferase reporter assay, bioinformatics and site-directed mutation results indicated that specificity protein 1 (SP1) and activator protein-1 (AP-1) motifs were located in the core region -1,063/-948, -606/-541 of the CYP1A1 promoter as well as -636/-595, -503/-462, -147/-1 of the CYP2A6 promoter. Furthermore, overexpression and decoy oligodeoxynucleotide technologies demonstrated that SP1 and AP-1 were pivotal transcriptional activators regulating the promoter activity of CYP1A1 and CYP2A6. Moreover, bioactivation of AFB1 to AFBO could be increased by upregulation of CYP1A1 and CYP2A6 expression, which was trans-activated owing to the upregulalion of AP-1, rather than SP1, stimulated by AFB1-induced reactive oxygen species. Additionally, nano-selenium could reduce ROS, downregulate AP-1 expression and then decrease the expression of CYP1A1 and CYP2A6, thus alleviating the toxicity of AFB1. In conclusion, AP-1 and SP1 played important roles in the transactivation of CYP1A1 and CYP2A6 expression and further bioactivated AFB1 to AFBO in chicken liver, which could provide novel targets for the remediation of aflatoxicosis in chicks.
Assuntos
Aflatoxina B1 , Galinhas , Citocromo P-450 CYP1A1 , Citocromo P-450 CYP2A6 , Fígado , Regiões Promotoras Genéticas , Fator de Transcrição Sp1 , Fator de Transcrição AP-1 , Animais , Aflatoxina B1/metabolismo , Galinhas/metabolismo , Fígado/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp1/genética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição AP-1/genética , Citocromo P-450 CYP2A6/metabolismo , Citocromo P-450 CYP2A6/genética , Ativação TranscricionalRESUMO
One of the members of CYP, a monooxygenase, CYP2A13 is involved in the metabolism of nicotine, coumarin, and tobacco-specific nitrosamine. Genetic polymorphisms have been identified in CYP2A13, with reported loss or reduction in enzymatic activity in CYP2A13 allelic variants. This study aimed to unravel the mechanism underlying the diminished enzymatic activity of CYP2A13 variants by investigating their three-dimensional structures through molecular dynamics (MD) simulations. For each variant, MD simulations of 1000 ns were performed, and the obtained results were compared with those of the wild type. The findings indicated alterations in the interaction with heme in CYP2A13.4, .6, .8, and .9. In the case of CYP2A13.5, observable effects on the helix structure related to the interaction with the redox partner were identified. These conformational changes were sufficient to cause a decrease in enzyme activity in the variants. Our findings provide valuable insights into the molecular mechanisms associated with the diminished activity in the CYP2A13 polymorphisms.
Assuntos
Simulação de Dinâmica Molecular , Nitrosaminas , Polimorfismo Genético , Nicotina , Oxirredução , Citocromo P-450 CYP2A6/genéticaRESUMO
Coumarin 7'-hydroxylase activity, a specific marker of CYP2A5 activity, and the protein level were measured in liver microsomes of male mice after chronic exposure to e-cigarettes (e-cigs) (2.4% nicotine). After exposure for 240 minutes per day for 5 days, the activity and the protein level in preproenkephalin (ppENK)-heterozygous [ppENK (+/-)] mice were significantly elevated (P <0.05) compared with the untreated control. This elevation was not due to deletion of the ppENK gene because the activity did not differ among untreated ppENK (+/-), ppENK (-/-), and wild-type ppENK (+/+) controls. Hence, the elevation can reasonably be attributed to nicotine exposure. The production of reactive oxygen species (ROS) upon incubation of the hepatic microsomes of these mice with cotinine was higher in microsomes from the e-cig-treated mice compared with the untreated controls (P < 0.01). Liquid chromatography mass spectrometry assay showed three oxidation products of cotinine, viz trans 3'-hydroxycotinine (3'-HC), 5'-hydroxycotinine (5'-HC), and cotinine N-oxide (CNO) in the plasma of these mice. The result identifies these three oxidation reactions as the source of the observed ROS and also shows that, in nicotine-treated mice, the appropriate "nicotine metabolite ratio" is (3'-HC + 5'-HC + CNO)/cotinine. The results suggest intriguing possibilities that 1) this metabolite ratio may correlate with plasma nicotine clearance and hence impact nicotine's psychoactive effects and 2) chronic e-cig treatment causes ROS-induced oxidative stress, which may play a major role in the regulation of CYP2A5 expression. Our present results clearly show that both the activity and the protein level of CYP2A5 are elevated by repeated exposure to nicotine. SIGNIFICANCE STATEMENT: Nicotine, the psychoactive ingredient of tobacco, is eliminated as the oxidation products of cotinine in reactions catalyzed by the enzymes CYP2A5 in mice and CYP2A6 in humans. This study shows that repeated exposure to e-cigarettes elevates the level of CYP2A5 and the formation of reactive oxygen species. The results suggest an intriguing possibility that CYP2A5 may be upregulated by chronic nicotine exposure due to oxidative stress caused by the oxidation of cotinine in this preclinical model of human smokers.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistemas Eletrônicos de Liberação de Nicotina , Masculino , Humanos , Animais , Camundongos , Cotinina/metabolismo , Nicotina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Microssomos Hepáticos/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2A6/metabolismoRESUMO
CYP2A6, a genetically variable enzyme, inactivates nicotine, activates carcinogens, and metabolizes many pharmaceuticals. Variation in CYP2A6 influences smoking behaviors and tobacco-related disease risk. This phenome-wide association study examined associations between a reconstructed version of our weighted genetic risk score (wGRS) for CYP2A6 activity with diseases in the UK Biobank (N = 395 887). Causal effects of phenotypic CYP2A6 activity (measured as the nicotine metabolite ratio: 3'-hydroxycotinine/cotinine) on the phenome-wide significant (PWS) signals were then estimated in two-sample Mendelian Randomization using the wGRS as the instrument. Time-to-diagnosis age was compared between faster versus slower CYP2A6 metabolizers for the PWS signals in survival analyses. In the total sample, six PWS signals were identified: two lung cancers and four obstructive respiratory diseases PheCodes, where faster CYP2A6 activity was associated with greater disease risk (Ps < 1 × 10-6). A significant CYP2A6-by-smoking status interaction was found (Psinteraction < 0.05); in current smokers, the same six PWS signals were found as identified in the total group, whereas no PWS signals were found in former or never smokers. In the total sample and current smokers, CYP2A6 activity causal estimates on the six PWS signals were significant in Mendelian Randomization (Ps < 5 × 10-5). Additionally, faster CYP2A6 metabolizer status was associated with younger age of disease diagnosis for the six PWS signals (Ps < 5 × 10-4, in current smokers). These findings support a role for faster CYP2A6 activity as a causal risk factor for lung cancers and obstructive respiratory diseases among current smokers, and a younger onset of these diseases. This research utilized the UK Biobank Resource.
Assuntos
Neoplasias Pulmonares , Doenças Respiratórias , Humanos , Nicotina/genética , Análise da Randomização Mendeliana , Fumar/efeitos adversos , Fumar/genética , Neoplasias Pulmonares/genética , Doenças Respiratórias/complicações , Citocromo P-450 CYP2A6/genética , Citocromo P-450 CYP2A6/metabolismoRESUMO
Genetic variation in CYP2B6 and CYP2A6 is known to impact interindividual response to antiretrovirals, nicotine, and bupropion, among other drugs. However, the full catalogue of clinically relevant pharmacogenetic variants in these genes is yet to be established, especially across African populations. This study therefore aimed to characterize the star allele (haplotype) distribution in CYP2B6 and CYP2A6 across diverse and understudied sub-Saharan African (SSA) populations. We called star alleles from 961 high-depth full genomes using StellarPGx, Aldy, and PyPGx. In addition, we performed CYP2B6 and CYP2A6 star allele frequency comparisons between SSA and other global biogeographical groups represented in the new 1000 Genomes Project high-coverage dataset (n = 2,000). This study presents frequency information for star alleles in CYP2B6 (e.g., *6 and *18; frequency of 21-47% and 2-19%, respectively) and CYP2A6 (e.g., *4, *9, and *17; frequency of 0-6%, 3-10%, and 6-20%, respectively), and predicted phenotypes (for CYP2B6), across various African populations. In addition, 50 potentially novel African-ancestry star alleles were computationally predicted by StellarPGx in CYP2B6 and CYP2A6 combined. For each of these genes, over 4% of the study participants had predicted novel star alleles. Three novel star alleles in CYP2A6 (*54, *55, and *56) and CYP2B6 apiece, and several suballeles were further validated via targeted Single-Molecule Real-Time resequencing. Our findings are important for informing the design of comprehensive pharmacogenetic testing platforms, and are highly relevant for personalized medicine strategies, especially relating to antiretroviral medication and smoking cessation treatment in Africa and the African diaspora. More broadly, this study highlights the importance of sampling diverse African ethnolinguistic groups for accurate characterization of the pharmacogene variation landscape across the continent.
Assuntos
Nicotina , Farmacogenética , Humanos , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2A6/genética , Frequência do Gene , África Subsaariana , Genótipo , AlelosRESUMO
CYP2A6 is a polymorphic enzyme that inactivates nicotine; structural variants (SVs) include gene deletions and hybrids with the neighboring pseudogene CYP2A7. Two studies found that CYP2A7 deletions were associated with ovarian cancer risk. Using their methodology, we aimed to characterize CYP2A6 SVs (which may be misidentified by prediction software as CYP2A7 SVs), then assess CYP2A6 SV-associated risk for ovarian cancer, and extend analyses to lung cancer. An updated reference panel was created to impute CYP2A6 SVs from UK Biobank array data. Logistic regression models analyzed the association between CYP2A6 SVs and cancer risk, adjusting for covariates. Software-predicted CYP2A7 deletions were concordant with known CYP2A6 SVs. Deleterious CYP2A6 SVs were not associated with ovarian cancer (OR = 1.06; 95% CI: 0.80-1.37; p = 0.7) but did reduce the risk of lung cancer (OR = 0.44; 95% CI: 0.29-0.64; p < 0.0001), and a lung cancer subtype. Replication of known lung cancer associations indicates the validity of array-based SV analyses.
Assuntos
Neoplasias Pulmonares , Neoplasias Ovarianas , Humanos , Feminino , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/genética , Bancos de Espécimes Biológicos , Biobanco do Reino Unido , Nicotina , Genótipo , Neoplasias Ovarianas/epidemiologia , Neoplasias Ovarianas/genética , Citocromo P-450 CYP2A6/genéticaRESUMO
Cigarette smoke, containing both nicotine and carcinogens, causes lung cancer. However, not all smokers develop lung cancer, highlighting the importance of the interaction between host susceptibility and environmental exposure in tumorigenesis. Here, we aimed to delineate the interaction between metabolizing ability of tobacco carcinogens and smoking intensity in mediating genetic susceptibility to smoking-related lung tumorigenesis. Single-variant and gene-based associations of 43 tobacco carcinogen-metabolizing genes with lung cancer were analyzed using summary statistics and individual-level genetic data, followed by causal inference of Mendelian randomization, mediation analysis, and structural equation modeling. Cigarette smoke-exposed cell models were used to detect gene expression patterns in relation to specific alleles. Data from the International Lung Cancer Consortium (29,266 cases and 56,450 controls) and UK Biobank (2,155 cases and 376,329 controls) indicated that the genetic variant rs56113850 C>T located in intron 4 of CYP2A6 was significantly associated with decreased lung cancer risk among smokers (OR = 0.88, 95% confidence interval = 0.85-0.91, P = 2.18 × 10-16), which might interact (Pinteraction = 0.028) with and partially be mediated (ORindirect = 0.987) by smoking status. Smoking intensity accounted for 82.3% of the effect of CYP2A6 activity on lung cancer risk but entirely mediated the genetic effect of rs56113850. Mechanistically, the rs56113850 T allele rescued the downregulation of CYP2A6 caused by cigarette smoke exposure, potentially through preferential recruitment of transcription factor helicase-like transcription factor. Together, this study provides additional insights into the interplay between host susceptibility and carcinogen exposure in smoking-related lung tumorigenesis. SIGNIFICANCE: The causal pathway connecting CYP2A6 genetic variability and activity, cigarette consumption, and lung cancer susceptibility in smokers highlights the need for behavior modification interventions based on host susceptibility for cancer prevention.
Assuntos
Neoplasias Pulmonares , Produtos do Tabaco , Humanos , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/genética , Citocromo P-450 CYP2A6/genética , Citocromo P-450 CYP2A6/metabolismo , Carcinógenos/toxicidade , Carcinogênese , Fatores de Transcrição , Fumar/efeitos adversosRESUMO
As a potential means for smoking cessation and consequently prevention of smoking-related diseases and mortality, in this study, our goal was to investigate the inhibition of nicotine metabolism by P450 2A6. Smoking is the main cause of many diseases and disabilities and harms nearly every organ of the body. As reported by the Centers for Disease Control and Prevention (CDC), more than 16 million Americans are living with diseases caused by smoking. On average, the life expectancy of a smoker is about 10 years less than a nonsmoker. Smoking cessation can substantially reduce the incidence of smoking-related diseases, including cancer. At least, 70 of the more than 7000 cigarette smoke components, including polycyclic aromatic hydrocarbons, N-nitrosamines, and aromatic amines, are known carcinogens. Nicotine is the compound responsible for the addictive and psychopharmacological effects of tobacco. Cytochrome P450 enzymes are responsible for the phase I metabolism of many tobacco components, including nicotine. Nicotine is mainly metabolized by cytochrome P450s 2A6 and 2A13 to cotinine. This metabolism decreases the amount of available nicotine in the bloodstream, leading to increased smoking behavior and thus exposure to tobacco toxicants and carcinogens. Here, we report the syntheses and P450 2A6 inhibitory activities of a number of new flavone-based esters and acids. Three of the flavone derivatives studied were found to be potent competitive inhibitors of the enzyme. Docking studies were used to determine the possible mechanisms of the activity of these inhibitors.
Assuntos
Flavonas , Nicotina , Humanos , Nicotina/farmacologia , Nicotina/metabolismo , Citocromo P-450 CYP2A6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Carcinógenos/metabolismo , Flavonas/farmacologiaRESUMO
BACKGROUND: Cigarette smoking is a modifiable risk factor associated with formation and rupture of intracranial aneurysms (IAs). Cytochrome P450 2A6 (CYP2A6) is the main enzyme implied in catabolism of nicotine and xenobiotics, giving rise to oxidative stress products. Our study investigated the associations between specific single-nucleotide polymorphisms (SNPs) in the CYP2A6 gene and the presence of sporadic IAs in a cluster of Italian patients, as well as their rupture regarding cigarette smoking habit. METHODS: Three hundred and thirty-one Italian patients with sporadic IAs were recruited in a single institution. We recorded data on clinical onset with subarachnoid hemorrhage (SAH) and smoking habit. Genetic analysis was performed with a standard procedure on peripheral blood samples: CYP2A6 ∗1B2, CYP2A6 ∗2, and CYP2A6 ∗14 SNPs were analyzed in the study group along with 150 healthy control subjects. Statistical analysis was conducted according to genetic association study guidelines. RESULTS: In the patient cohort, the frequency of aSAH was significantly higher in current smokers (P < 0.001; OR=17.45), regardless of the pattern of CYP2A6 SNPs. There was a correlation between IA rupture and cigarette smoking in patients with the heterozygous CYP2A6 ∗1B2 allele (P < 0.001; OR=15.47). All patients carrying the heterozygous CYP2A6 ∗14 allele had an aSAH event (100%), regardless of smoking habit, although this correlation was not statistically significant (P = 1). CONCLUSIONS: According to our findings, a cigarette smoker carrying a fully active CYP2A6 enzyme (heterozygous ∗1B2 allele) may have an increased risk of IA rupture compared to those with functionally less active variants: further investigation on a larger sample is needed to verify this result. The role of the heterozygous CYP2A6 ∗14 allele in aSAH is yet to be clarified.
Assuntos
Fumar Cigarros , Aneurisma Intracraniano , Humanos , Polimorfismo de Nucleotídeo Único/genética , Fumar Cigarros/efeitos adversos , Fumar Cigarros/epidemiologia , Fumar Cigarros/genética , Aneurisma Intracraniano/epidemiologia , Aneurisma Intracraniano/genética , Fatores de Risco , Itália/epidemiologia , Citocromo P-450 CYP2A6/genéticaRESUMO
CYP2A6 metabolically inactivates nicotine. Faster CYP2A6 activity is associated with heavier smoking and higher lung cancer risk. The CYP2A6 gene is polymorphic, including functional structural variants (SV) such as gene deletions (CYP2A6*4), duplications (CYP2A6*1 × 2), and hybrids with the CYP2A7 pseudogene (CYP2A6*12, CYP2A6*34). SVs are challenging to genotype due to their complex genetic architecture. Our aims were to develop a reliable protocol for SV genotyping, functionally phenotype known and novel SVs, and investigate the feasibility of CYP2A6 SV imputation from SNP array data in two ancestry populations. European- (EUR; n = 935) and African- (AFR; n = 964) ancestry individuals from smoking cessation trials were genotyped for SNPs using an Illumina array and for CYP2A6 SVs using Taqman copy number (CN) assays. SV-specific PCR amplification and Sanger sequencing was used to characterize a novel SV. Individuals with SVs were phenotyped using the nicotine metabolite ratio, a biomarker of CYP2A6 activity. SV diplotype and SNP array data were integrated and phased to generate ancestry-specific SV reference panels. Leave-one-out cross-validation was used to investigate the feasibility of CYP2A6 SV imputation. A minimal protocol requiring three Taqman CN assays for CYP2A6 SV genotyping was developed and known SV associations with activity were replicated. The first domain swap CYP2A6-CYP2A7 hybrid SV, CYP2A6*53, was identified, sequenced, and associated with lower CYP2A6 activity. In both EURs and AFRs, most SV alleles were identified using imputation (>70% and >60%, respectively); importantly, false positive rates were <1%. These results confirm that CYP2A6 SV imputation can identify most SV alleles, including a novel SV.
Assuntos
População Africana , População Europeia , Nicotina , Abandono do Hábito de Fumar , Humanos , População Africana/genética , Sequência de Bases , População Negra/genética , Citocromo P-450 CYP2A6/genética , Citocromo P-450 CYP2A6/metabolismo , População Europeia/genética , Genótipo , Nicotina/genética , Nicotina/metabolismo , Polimorfismo de Nucleotídeo Único , População Branca/genética , Abandono do Hábito de Fumar/etnologiaRESUMO
Biphasic hyalinizing psammomatous renal cell carcinoma (BHP RCC) with NF2 gene mutations is a newly described provisional category of renal cell carcinoma (RCC). Here we described three additional cases of BHP RCC with CYP2A6 gene mutation besides NF2 gene. The carcinomas were predominantly unencapsulated, and two of them had a rounded, nodular interface with the native kidney while one had perirenal adipose tissue invasion. Histopathologically, all neoplasms had a characteristic biphasic appearance of smaller cells clustering around basement membrane material within larger acini, forming pseudorosettes or a glomeruloid pattern. The smaller cells were focally spindle-shaped in two carcinomas. Psammoma bodies were shown in two carcinomas. Cellular necrosis and perineural invasion was identified in one case. Immunohistochemically, Vimentin, EMA, P504s were extensively expressed while RCC and CD10 were only expressed in larger cells. CK7 was positive in one tumor. CYP2A6 gene mutation (CYP2A6 NM_000762.6: exon4:c.A580G:p.K194E) was revealed in three tumors by Whole-genome exome sequencing, which was further conï¬rmed by Sanger sequencing. Only one case harbored a somatic termination mutation in NF2 gene. NF2 promoter methylation was observed in the other two cases. Clinically, one patient died of disease with widespread bone metastases confirmed by biopsy at the ninth month after surgery but the other two patients had no evidence of recurrence or metastases (follow-up period 9-90 months). Our ï¬ndings validated previously described clinicopathological features and NF2 gene mutation or promoter methylation of BHP RCC. In addition, we reported different IHC pattern of BHP RCC and further revealed the recurrent CYP2A6 genetic alteration.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Neoplasias Meníngeas , Meningioma , Humanos , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Biomarcadores Tumorais/genética , Mutação , Citocromo P-450 CYP2A6/genéticaRESUMO
Cytochrome P450 2A6 (CYP2A6) inhibitors are expected to be suitable as smoking cessation aids and for cancer prevention. Because the typical coumarin-based CYP2A6 inhibitor methoxsalen also inhibits CYP3A4, unintended drug-drug interactions are still a concern. Therefore, the development of selective CYP2A6 inhibitors is desirable. In this study, we synthesized coumarin-based molecules, determined the IC50 values for CYP2A6 inhibition, verified the possibility of mechanism-based inhibition, and compared the selectivity for CYP2A6 versus CYP3A4. The results demonstrated that we developed CYP2A6 inhibitors that were more potent and selective than methoxsalen.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Inibidores das Enzimas do Citocromo P-450/farmacologia , Citocromo P-450 CYP3A , Metoxaleno/farmacologia , Cumarínicos/farmacologia , Citocromo P-450 CYP2A6 , Microssomos HepáticosRESUMO
INTRODUCTION: Genetic variation in Cytochrome P450 2A6 (CYP2A6), the major nicotine metabolizing enzyme, is associated with nicotine dependence and smoking cessation. Nicotine dependence severity also predicts smoking cessation. Our goals were to determine how CYP2A6 variation and nicotine dependence alter smoking cessation, and whether dependence could refine CYP2A6-based treatment recommendations. AIMS AND METHODS: Adult smokers treated for 12 weeks with placebo, nicotine patch, or varenicline (NCT01314001) were grouped as CYP2A6 normal (n = 567) or slow (n = 432) nicotine metabolizers based on a CYP2A6 weighted genetic risk score. Fagerström test for nicotine dependence scores were measured at baseline and biochemically verified smoking cessation was assessed at end of treatment. RESULTS: Dependence neither mediated nor moderated an association between CYP2A6 variation and smoking cessation overall, within any treatment arm, or after stratifying by ancestry (n = 591 European, n = 408 African ancestry) or sex (n = 444 women, n = 555 men). In within-treatment analyses, the mediation effect odds ratio (OR) ranged from 0.95 to 1.00 and the bias-corrected 95% confidence interval contained 1. Moderation (i.e. interaction) effect ORs ranged from 0.88 to 1.61 (p = .397-.828). For CYP2A6 normal metabolizers, quit rates on varenicline were similar for those with high (41.1%) and low (43.4%) dependence, while quit rates were lower for those with high versus low dependence on both patch (16.5 vs. 29.7%) and placebo (8.9 vs. 18.5%). CYP2A6 slow metabolizers with high versus low dependence had lower quit rates in all three treatment arms. CONCLUSIONS: Although nicotine dependence severity neither mediated nor moderated CYP2A6 associations with smoking cessation, incorporating information on dependence may optimize the choice of smoking cessation treatment aid in CYP2A6 normal and slow metabolizers. IMPLICATIONS: Variation in CYP2A6 and nicotine dependence severity alter smoking cessation success. Our findings suggest that while nicotine dependence severity is unlikely to mediate or moderate CYP2A6 associations with cessation, incorporating patient information on both CYP2A6 and nicotine dependence severity may lead to improved smoking cessation strategies.
Assuntos
Abandono do Hábito de Fumar , Tabagismo , Adulto , Feminino , Humanos , Masculino , Citocromo P-450 CYP2A6/genética , Variação Genética , Nicotina/uso terapêutico , Tabagismo/terapia , Tabagismo/tratamento farmacológico , Vareniclina/uso terapêuticoRESUMO
Smoking intensity varies across smokers and is influenced by individual variability in the metabolism of nicotine, the major addictive agent in tobacco. Therefore, lung cancer risk, which varies by racial ethnic group, is influenced by the primary catalyst of nicotine metabolism, cytochrome P450 2A6 (CYP2A6). In smokers, CYP2A6 catalyzes nicotine 5'-oxidation. In vitro, CYP2A6 also catalyzes, to a much lower extent, 2'-oxidation, which leads to the formation of 4-hydroxy-4-(3-pyridyl) butanoic acid (hydroxy acid). The urinary concentration of hydroxy acid has been quantified in only a few small studies of White smokers. To quantitatively assess the importance of nicotine 2'-oxidation in smokers, an LC-MS/MS-based method was developed for the analysis of nicotine and ten metabolites in urine. The concentrations of nicotine and these metabolites were measured in 303 smokers (99 Whites, 99 Native Hawaiians, and 105 Japanese Americans), and the relative metabolism of nicotine by four pathways was determined. Metabolism by these pathways was also compared across quartiles of CYP2A6 activity (measured as the plasma ratio of 3-hydroxycotinine to cotinine). As reported previously and consistent with their average CYP2A6 activity, nicotine 5'-oxidation was highest in Whites and lowest in Japanese Americans. Nicotine N-glucuronidation and N-oxidation increased with decreasing CYP2A6 activity. However, the relative urinary concentration of hydroxy acid (mean, 2.3%; 95% CI, 2.2-2.4%) did not vary by ethnic group or by CYP2A6 activity. In summary, CYP2A6 is not an important catalyst of nicotine 2'-oxidation in smokers, nor does nicotine 2'-oxidation compensate for decreased CYP2A6 activity.
Assuntos
Asiático , Nicotina , Humanos , Nicotina/metabolismo , Ácido Butírico , Havaiano Nativo ou Outro Ilhéu do Pacífico , Cromatografia Líquida , Brancos , Espectrometria de Massas em Tandem , Cotinina/metabolismo , Citocromo P-450 CYP2A6RESUMO
Cannabis-based products have experienced notable increases in co-usage alongside tobacco products. Several cannabinoids exhibit inhibition of a number of cytochrome P450 (CYP) and UDP glucuronosyltransferase (UGT) enzymes, but few studies have examined their inhibition of enzymes involved in nicotine metabolism. The goal of the present study was to examine potential drug-drug interactions occurring in the nicotine metabolism pathway perpetrated by cannabidiol (CBD) and its active metabolite, 7-hydroxy-CBD (7-OH-CBD). The inhibitory effects of CBD and 7-OH-CBD were tested in microsomes from HEK293 cells overexpressing individual metabolizing enzymes and from human liver tissue. Assays with overexpressing microsomes demonstrated that CBD and 7-OH-CBD inhibited CYP-mediated nicotine metabolism. Binding-corrected IC50,u values for CBD inhibition of nicotine metabolism to cotinine and nornicotine, and cotinine metabolism to trans-3'-hydroxycotinine (3HC), were 0.27 ± 0.060, 0.23 ± 0.14, and 0.21 ± 0.14 µM, respectively, for CYP2A6; and 0.26 ± 0.17 and 0.029 ± 0.0050 µM for cotinine and nornicotine formation, respectively, for CYP2B6. 7-OH-CBD IC50,u values were 0.45 ± 0.18, 0.16 ± 0.08, and 0.78 ± 0.23 µM for cotinine, nornicotine, and 3HC formation, respectively, for CYP2A6, and 1.2 ± 0.44 and 0.11 ± 0.030 µM for cotinine and nornicotine formation, respectively, for CYP2B6. Similar IC50,u values were observed in HLM. Inhibition (IC50,u = 0.37 ± 0.06 µM) of 3HC to 3HC-glucuronide formation by UGT1A9 was demonstrated by CBD. Significant inhibition of nicotine metabolism pathways by CBD and 7-OH-CBD suggests that cannabinoids may inhibit nicotine metabolism, potentially impacting tobacco addiction and cessation.
Assuntos
Canabidiol , Canabinoides , Nicotina , Humanos , Canabidiol/farmacologia , Canabinoides/metabolismo , Canabinoides/farmacologia , Cotinina/metabolismo , Citocromo P-450 CYP2A6/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Células HEK293 , Microssomos Hepáticos/metabolismo , Nicotina/farmacologia , Nicotina/metabolismoRESUMO
The tree shrew, a non-rodent primate-like species, is used in various fields of biomedical research, including hepatitis virus infection, myopia, depression, and toxicology. Recent genome analysis found that the numbers of cytochrome P450 (P450 or CYP) genes are similar in tree shrews and humans and their sequence identities are high. Although the P450s are a family of important drug-metabolizing enzymes, they have not yet been fully investigated in tree shrews. In the current study, tree shrew CYP2A13 cDNA was isolated from liver, and its characteristics were compared with those of pig, dog, and human CYP2As. Tree shrew CYP2A13 amino acid sequences were highly identical (87-92%) to the human CYP2As and contained sequence motifs characteristic of P450s. Phylogenetic analysis revealed that tree shrew CYP2A13 was more closely related to human CYP2As than to rat CYP2As, similar to dog and pig CYP2As. Among the tissue types analyzed, tree shrew CYP2A13 mRNA was preferentially expressed in liver and lung, similar to dog CYP2A13 mRNA, whereas dog CYP2A25 and pig CYP2A19 mRNAs were predominantly expressed in liver. Tree shrew liver microsomes and tree shrew CYP2A13 proteins heterologously expressed in Escherichia coli catalyzed coumarin 7-hydroxylation and phenacetin O-deethylation, just as human, dog, and pig CYP2A proteins and liver microsomes do. These results demonstrate that tree shrew CYP2A13 is expressed in liver and lung and encodes a functional drug-metabolizing enzyme. SIGNIFICANCE STATEMENT: Novel tree shrew cytochrome P450 2A13 (CYP2A13) was identified and characterized in comparison with human, dog, and pig CYP2As. Tree shrew CYP2A13 isolated from liver had high sequence identities and close phylogenetic relationships to its human homologs and was abundantly expressed in liver and lung at the mRNA level. Tree shrew CYP2A13 metabolized coumarin and phenacetin, human selective CYP2A6 and CYP2A13 substrates, respectively, similar to dog and pig CYP2As, and is a functional drug-metabolizing enzyme likely responsible for drug clearances.
Assuntos
Sistema Enzimático do Citocromo P-450 , Tupaia , Animais , Cães , Humanos , Ratos , Citocromo P-450 CYP2A6/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Fenacetina , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Tupaia/genética , Tupaia/metabolismoRESUMO
CYP2A6 is a very important enzyme that plays a crucial role in nicotine compounds and is responsible for the metabolism of more than 3% drugs of total metabolized drugs by the CYP family and reported as one of very important pharmacogenes. CYP2A6 is highly polymorphic in nature and reported with more than 40 variants, most of these variants are SNPs originated and population specific. It has been well observed and reported that the presence of these population-specific non-synonymous SNPs in CYP2A6 alters the rate of drug metabolism and as a functional consequence, drugs produce an abnormal response. Though genomics and pharmacogenomics studies are there, very less is known about the structural effects of these SNPs on molecular-interaction and folding of CYP2A6. To fill the knowledge gap, SNPs based four variants, i.e., CYP2A6*2, CYP2A6*18, CYP2A6*21, and CYP2A6*35, which are frequently reported in the South Asian population, were considered for the study. Coumarin (DB04665), a well reported drug, is considered as a model substance, and the effect of all four variants on 'CYP2A6*-coumarin' complex was studied. MD simulation-based analysis (at 200 ns) was performed and comparative analysis with respect to wild type 'CYP2A6-coumarin' complex was done. Though observation didn't find any global effect on complete complex but found some crucial minor-local alteration in interaction and folding process. It is assumed that the change due to SNPs in the single amino acid did not bring global change in physiochemical properties of CYP2A6* but caused local-trivial changes which are very crucial for its metabolic activity.Communicated by Ramaswamy H. Sarma.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Citocromo P-450 CYP2A6 , Oxigenases de Função Mista , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/metabolismo , Cumarínicos , Citocromo P-450 CYP2A6/genética , Citocromo P-450 CYP2A6/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Simulação de Dinâmica Molecular , Nicotina/metabolismoRESUMO
BACKGROUND: Nicotine metabolism is a major factor in nicotine dependence, with approximately 70% to 80% of nicotine metabolized to cotinine in Caucasians. Cotinine formation is catalyzed primarily by CYP2A6, which also converts cotinine to trans-3'-hydroxycotinine (3HC). The goal of the present study was to examine the effects of CYP2A6 deficiency on nicotine metabolism profiles in vivo and the importance of genetic variants in nicotine-metabolizing enzyme genes on urinary nicotine metabolites levels. METHODS: Urine samples from 722 smokers who participated in the Singapore Chinese Health Study were analyzed using UPLC-MS/MS to detect nicotine and eight of its urinary metabolites, and a total of 58 variants in 12 genes involved in nicotine metabolism were investigated in 475 of these subjects with informative genotyping data. RESULTS: Urine samples stratified by the ratio of 3HC/cotinine exhibited a 7-fold increase in nicotine-N'-oxide, a 6-fold increase in nicotine-Glucuronide (Gluc), and a 5-fold decrease in 3HC-Gluc when comparing the lower versus upper 3HC/cotinine ventiles. Significant (P < 0.0001) associations were observed between functional metabolizing enzyme genotypes and levels of various urinary nicotine metabolites, including CYP2A6 genotype and levels of nicotine, nicotine-Gluc, nicotine-N'-oxide and 3HC, UGT2B10 genotype and levels of cotinine, nicotine-Gluc and cotinine-Gluc, UGT2B17 genotype and levels of 3HC-Gluc, FMO3 genotype and levels of nicotine-N'-oxide, and CYP2B6 genotype and levels of nicotine-N'-oxide and 4-hydroxy-4-(3-pyridyl)-butanoic acid. CONCLUSIONS: These data suggest that several pathways are important in nicotine metabolism. IMPACT: Genotype differences in several nicotine-metabolizing enzyme pathways may potentially lead to differences in nicotine dependence and smoking behavior and cessation.