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.

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.

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 confirmed 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 findings 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.

Deferiprone (DFP) is a metal chelating agent generally used to treat patients with thalassaemia, due to iron overload in clinical settings.Studies have revealed that long-term use of DFP can induce hepatotoxicity, however, mechanisms of its toxic action remain unclear. The present studies are aimed to characterize the reactive metabolite of DFP, to define the metabolic pathway, and to determine the P450 enzymes participating in the bioactivation.A demethylation metabolite (M1) was observed in rat liver microsomal incubations. Additionally, a glutathione (GSH) conjugate (M2) and an N-acetylcysteine (NAC) conjugate (M3) were detected in microsomal incubations fortified with DFP and GSH/NAC.Biliary M2 and urinary M3 were respectively found in animals administered DFP.CYP2A6 enzyme dominated the catalysis to bioactivate DFP.

Unraveling the molecular mechanisms by which genetic variants of cytochrome P450 2A6 lead to different metabolic phenotypes remains a long-standing but important challenge. CYP2A6 is an enzyme involved in the metabolism of several clinical drugs as well as the metabolic activation of carcinogenic nitrosamines. Herein, CYP2A6 genotypes and phenotypes, as indicated by protein content [by liquid chromatography-mass spectrometry (MS)/MS] and metabolic activities [Vmax, clearance (CL)], were determined for 90 human liver samples. We determined the median, range, and interindividual and intraindividual variation of CYP2A6 content and activity at the microsomal, liver tissue, and whole liver level and predicted hepatic in vivo clearance by in vitro-in vivo extrapolation based on CYP2A6-mediated coumarin metabolism by each CYP2A6 genotype. These results reveal how different CYP2A6 genotypes yield different phenotypic traits in protein content and enzyme activity. For relative Vmax, CL, and protein content, the intraindividual percentage coefficients of variation (ICVs) were 41.0% (18.8%-125.1%), 28.5% (2.39%-133.5%), and 27.8% (2.68%-88.0%), respectively. The high ICVs implied large intraindividual variation at different levels, sometimes in a genotype-dependent manner. Intergenotype analysis revealed that the CYP2A6*4 allele demonstrated the most obvious effect on phenotypic outcomes, both in protein content and in metabolic activity. Indeed, decreased CYP2A6 protein content with the CYP2A6*4 genotype might explain the decreased metabolic activity from the molecular to the organismal level. These findings may allow useful predictions for CYP2A6-mediated drug metabolism on an individual patient basis in accord with the goal of achieving personalized medicine. SIGNIFICANCE STATEMENT: We provide the median, range, and interindividual and intraindividual variation in CYP2A6 content at the microsomal, liver tissue, and whole liver level by liquid chromatography-mass spectrometry (MS)/MS as well as activities at the protein, microsomal, liver tissue, and whole liver level both in vitro and at the organismal level based on CYP2A6-mediated coumarin metabolism with each CYP2A6 genotype, thereby allowing us to elucidate how different CYP2A6 genotypes yield differing phenotypic traits (protein content and enzyme activity), facilitating the development of personalized medicine.

Dexmedetomidine is an important sedative agent administered as premedication to achieve procedural sedation in children. To describe the correlation between the genetic state and the concentration of dexmedetomidine, it is necessary to develop a specific, time-saving and economical method for detection of dexmedetomidine in plasma samples. In this work, an ultra-high-performance liquid chromatography (UHPLC)-tandem mass spectrometry method has been established and validated for detection of dexmedetomidine in plasma from pediatric population. After a simple liquid-liquid extraction with an organic solution, the analytes were separated on an ACQUITY BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) by gradient elution with the mobile phase of acetonitrile and 1‰ aqueous formic acid (flow rate 0.3 mL min-1 ). Mass spectrometry measurements were performed under the positive selected reaction monitoring and the mass transitions monitored were m/z 201.3 → 95.1, 204.2 → 98.0 for dexmedetomidine and deuterated medetomidine (internal standard), respectively. Validation of the method based on China Food and Drug Administration guidelines showed acceptable selectivity. The UHPLC method employed a stable isotope-labeled internal standard, showed good specificity and was successfully used to detect dexmedetomidine in plasma samples from 260 pediatric patients. A subsequent application of this method to a pharmacogenetic study was also described. Importantly, this is the first study to report the correlation between CYP2A6 rs835309 activity and concentration of dexmedetomidine.

Compared with coumarin, 7-hydroxycoumarin could serve as a better hit for developing CYP2A6 inhibitors. In this study, a series of 7-hydroxycoumarin and its structural analogues were collected to study their structure-activity relationship (SAR) and isoform selectivity for inhibiting CYP2A6. All tested coumarins except a C4 phenyl derivative (11) showed higher inhibitory activities for CYP2A6 over the other CYP isoforms, including CYP1A2, CYP2D6, CYP2E1, CYP3A4, CYP2C8, and CYP2C9. Of these coumarins, 6,7-dihydroxycoumarin (1) and 7,8-dihydroxycoumarin (9) were found to be potent inhibitors of CYP2A6 with IC50/Ki value of 0.39/0.25 and 4.61/3.02 μM, respectively, compared to methoxalen as positive control (IC50/Ki = 0.43/0.26 μM). In contrast, other coumarins showed low or decreased CYP2A6-inhibiting activities. SAR analysis showed that hydroxy groups might be important for CYP2A6 inhibition, and the rank order of sites for hydroxy substitution was C6 > C7 > C8. In addition, either hydrophobic or hydrophilic substituents introduced into C4, C6 and C8 led to a reduction in CYP2A6-inhibiting activity, and the degree of influence was dependent on the size and electrical charge of substituents. Furthermore, inhibition kinetic analysis and docking simulations demonstrated that the 8-O-glucosylated coumarin derivative (17) exhibited noncompetitive inhibition against CYP2A6, while competitive inhibition patterns were noted for the other tested coumarins. The mechanisms underlying the inhibitors binding to CYP2A6 were further investigated by molecular docking study. The findings presented herein are very helpful for developing highly selective and more potent CYP2A6 inhibitors.

The aim of this study was to evaluate the association between CYP2A13 polymorphisms and lung cancer susceptibility using the HapMap database.
A case-control analysis of 532 subjects with lung cancer and 614 controls with no personal history of the disease was performed. The tag SNPs rs1645690 and rs8192789 for CYP2A13 were selected, and the genetic polymorphisms were confirmed experimentally through real-time PCR, cloning, and sequencing assay.
SNP frequency in this study was consistent with the HapMap Project database of Han-Chinese and lung cancer risk was associated with CYP2A13 polymorphisms in non-smokers. CYP2A13 shares a 93.5% identity with CYP2A6 in the amino acid sequence and the homologous sequences may interfere with the study of SNPs of CYP2A13.
CYP2A13 may be a potential key metabolic enzyme gene in the carcinogenesis of lung cancer in non-smokers. The common polymorphisms of CYP2A13 may be candidate biomarkers for lung cancer susceptibility in Han-Chinese.

The major mode of metabolism of nicotine is by hydroxylation via cytochrome P450 (CYP) 2A6, but it can also undergo glucuronidation by UDP-glucuronosyltransferases and oxidation by flavin monooxygenases (FMO). The goal of this study was to examine the potential importance of FMOs in nicotine metabolism and assess the potential impact of missense polymorphisms in active FMOs on nicotine-N\'-oxide (NOX) formation.
Urine samples from 106 current Chinese smokers were analyzed for nicotine metabolites by mass spectrometry. Wild-type FMOs 1-5 and their most prevalent nonsynonymous variants were cloned and overexpressed in HEK293 cells, and were tested in oxidation reactions against nicotine.
A strong inverse correlation was observed between the ratio of urinary 3\'-hydroxycotinine/cotinine, a measure of CYP2A6 activity, and the urinary levels of NOX alone (r = -0.383; P < 0.001) or NOX measured as a ratio of total nicotine metabolites (r = -0.414; P < 0.001) in smokers. In addition to FMO1 and FMO3, the functional FMO2427Q isoform was active against nicotine, whereas FMO4 and FMO5 exhibited low activity against nicotine (K m > 5.0 mmol/L). Significant (P < 0.05) decreases in N\'-oxidation activity (V max/K m) were observed for the FMO1I303V, FMO3N61S, FMO3D132H, FMO3V257M, and FMO3E308G variants in vitro when compared with their respective wild-type isoforms; the truncated FMO2Q472stop isoform exhibited no enzyme activity.
These data indicate that increases in nicotine-N\'-oxidation occur in subjects with deficient CYP2A6 activity, and that several FMO enzymes are active in nicotine-N\'-oxidation.
Several common missense FMO variants are associated with altered enzyme activity against nicotine and may play an important role in nicotine metabolism in low-CYP2A6 activity subjects.

OBJECTIVE: This study aimed to evaluate the links between CYP450 family genes in tumor tissues and hepatocellular carcinoma (HCC) outcomes.
METHODS: Gene Expression Omnibus (GEO) databases GSE14520 and GSE36376 were used to identify differential expressed CYP450 genes between tumor and nontumor tissues and related to HCC clinicopathological features and survivals.
RESULTS: Seven CYP450 genes including CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2E1, CYP3A4, and CYP4A11 were downregulated in tumor tissues, which were validated in both GSE14520 and GSE36376. HCC patients with CYP2A6 and CYP2C8 low levels in tumor tissues suffered from poorer overall survival (OS) compared to those with high CYP2A6 and CYP2C8 in GSE14520 profile (log ranks P = 0.01 and P = 0.006, respectively). In addition, HCC patients with lower CYP2A6 and CYP2C8 in tumors had worse recurrence-free survival (RFS) than those with higher CYP2A6 and CYP2C8 (log ranks P = 0.02 and P = 0.012, respectively). In GSE36376 validation dataset, HCC patients with lower CYP2A6 and CYP2C8 had worse OS and RFS than those with higher CYP2A6 and CYP2C8 (all P < 0.05), in line with results in GSE14520 dataset. Additionally, lower CYP2A6 and CYP2C8 are associated with advanced clinicopathological features including tumor staging, vascular invasion, intrahepatic metastasis, and high alpha fetoprotein (all P < 0.05).
CONCLUSIONS: Downregulation of CYP2A6 and CYP2C8 in tumor tissues links to poorer OS and RFS in HCC patients.