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.

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.

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.

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.

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.

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.
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.

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.

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.

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.

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.