Targeting estrogen mediated CYP4F2/CYP4F11-20-HETE metabolic disorder decelerates tumorigenesis in ER+ breast cancer.

Purpose: As the most common subset of breast cancer (BC), estrogen receptor positive (ER+) BC accounting for 80% of cases, has become a global public health concern. The female hormone estrogen (E2) unequivocally drives ER + breast malignancies. The reasons that estrogen affects BC development has long been considered, yet further study remains to be conducted of the molecular events in the E2-estrogen receptor α (ERα) signaling pathway in ER + BC progression, especially lipid metabolism, so providing more options for tailored and individualized therapy. Our aim is to find out new targets and clinical biomarkers for ER + breast cancer treatment from the perspective of lipid metabolism. Methods: Lipid metabolomics profiling was used to examine the membrane phospholipid stimulated by E2. Clinical BC samples were used to assess the association of CYP4F2, CYP4F11 expression with clinicopathological characteristics and patient outcomes. Some inhibitors of main enzymes in AA metabolism were used combined with E2 to assess roles of CYP4F2/CYP4F11 in the progression of ER + BC. CYP4F2, CYP4F11 overexpression and knockdown BC cell lines were employed to examine the effects of CYP4F2, CYP4F11 on cellular proliferation, apoptosis and tumor growth. Western blotting, qPCR, Immunohistochemical staining and flow cytometry were also conducted to determine the underlying mechanisms related to CYP4F2, CYP4F11 function. Results: The activation of the CYP450 signaling pathway in arachidonic acid metabolism contributed to ER + BC tumorigenesis. In ER + BC, CYP4F2 and CYP4F11 overexpression induced by E2 could promote cancer cell proliferation and resistance to apoptosis by producing the metabolite 20-HETE and activating the antiapoptotic protein Bcl-2. CYP4F2 and CYP4F11 elevation correlates with poorer overall survival and disease-free survival in ER + BC patients. Conclusion: CYP4F2, CYP4F11 and their metabolite 20-HETE could serve as effective prognostic markers and attractive therapeutic targets for novel anticancer drug development about ER + BC.

Genetic Variants of CYP4F2 Associated with Ischemic Stroke Susceptibility in the Han Population from Southern China.

Background: The pathophysiological mechanism of ischemic stroke is complex. Traditional risk factors cannot fully or only partially explain the occurrence and development of IS. Genetic factors are getting more and more attention. Our study aimed to explore the association between CYP4F2 gene polymorphism and susceptibility to IS. Methods: A total of 1322 volunteers were enrolled to perform an association analysis through SNPStats online software. Using FPRP (false-positive report probability) to detect whether the result is a noteworthy finding. The interaction of SNP-SNP in IS risk was assessed by multi-factor dimensionality reduction. Statistical analysis of this study was mainly completed by SPSS 22.0 software. Results: Mutant allele "A" (OR = 1.24) and genotype "AA" (OR = 1.49) or "GA" (OR = 1.26) of CYP4F2-rs2108622 are risk genetic factors for IS. Rs2108622 is significantly associated with an increased risk of IS among subjects who are females, aging >60 years old, with BMI ≥24 kg/m2, and smoking or drinking volunteers. CYP4F2-rs3093106 and -rs3093105 are associated with susceptibility to IS among smoking, drinking subjects, or IS patients complicated with hypertension. Conclusion: CYP4F2-rs2108622, -rs3093106, and -rs3093105 are associated with an increased risk of IS.

Genetic polymorphisms in CYP4F2 may be associated with lung cancer risk among females and no-smoking Chinese population.

Background: Our study aimed to explore the potential association of CYP4F2 gene polymorphisms with lung cancer (LC) risk. Methods: The five variants in CYP4F2 were genotyped using Agena MassARRAY in 507 cases and 505 controls. Genetic models and haplotypes based on logistic regression analysis were used to evaluate the potential association between CYP4F2 polymorphisms and LC susceptibility. Results: This study observed that rs12459936 was linked to an increased risk of LC in no-smoking participants (allele: OR = 1.38, p = 0.035; homozygote: OR = 2.00, p = 0.035; additive: OR = 1.40, p = 0.034) and females (allele: OR = 1.64, p = 0.002; homozygote: OR = 2.57, p = 0.006; heterozygous: OR = 2.56, p = 0.001; dominant: OR = 2.56, p < 0.002; additive: OR = 1.67, p = 0.002). Adversely, there was a significantly decreased LC risk for rs3093110 in no-smoking participants (heterozygous: OR = 0.56, p = 0.027; dominant: OR = 0.58, p = 0.035), rs3093193 (allele: OR = 0.66, p = 0.016; homozygote: OR = 0.33, p = 0.011; recessive: OR = 0.38, p = 0.021; additive: OR = 0.64, p = 0.014), rs3093144 (recessive: OR = 0.20, p = 0.045), and rs3093110 (allele: OR = 0.54, p = 0.010; heterozygous: OR = 0.50, p = 0.014; dominant: OR = 0.49, p = 0.010; additive: OR = 0.54, p = 0.011) in females. Conclusions: The study demonstrated that CYP4F2 variants were associated with LC susceptibility, with evidence suggesting that this connection may be affected by gender and smoking status.

Change in plasma α-tocopherol associations with attenuated pulmonary function decline and with CYP4F2 missense variation.

BACKGROUND: Vitamin E (vitE) is hypothesized to attenuate age-related decline in pulmonary function. OBJECTIVES: We investigated the association between change in plasma vitE (∆vitE) and pulmonary function decline [forced expiratory volume in the first second (FEV1)] and examined genetic and nongenetic factors associated with ∆vitE. METHODS: We studied 1144 men randomly assigned to vitE in SELECT (Selenium and Vitamin E Cancer Prevention Trial). ∆vitE was the difference between baseline and year 3 vitE concentrations measured with GC-MS. FEV1 was measured longitudinally by spirometry. We genotyped 555 men (vitE-only arm) using the Illumina Expanded Multi-Ethnic Genotyping Array (MEGAex). We used mixed-effects linear regression modeling to examine the ∆vitE-FEV1 association. RESULTS: Higher ∆vitE was associated with lower baseline α-tocopherol (α-TOH), higher baseline γ-tocopherol, higher baseline free cholesterol, European ancestry (as opposed to African) (all P < 0.05), and the minor allele of a missense variant in cytochrome P450 family 4 subfamily F member 2 (CYP4F2) (rs2108622-T; 2.4 µmol/L higher ∆vitE, SE: 0.8 µmol/L; P = 0.0032). Higher ∆vitE was associated with attenuated FEV1 decline, with stronger effects in adherent participants (≥80% of supplements consumed): a statistically significant ∆vitE × time interaction (P = 0.014) indicated that a 1-unit increase in ∆vitE was associated with a 2.2-mL/y attenuation in FEV1 decline (SE: 0.9 mL/y). The effect size for 1 SD higher ∆vitE (+4 µmol/mmol free-cholesterol-adjusted α-TOH) was roughly one-quarter of the effect of 1 y of aging, but in the opposite direction. The ∆vitE-FEV1 association was similar in never smokers (2.4-mL/y attenuated FEV1 decline, SE: 1.0 mL/y; P = 0.017, n = 364), and current smokers (2.8-mL/y, SE: 1.6 mL/y; P = 0.079, n = 214), but there was little to no effect in former smokers (-0.64-mL/y, SE: 0.9 mL/y; P = 0.45, n = 564). CONCLUSIONS: Greater response to vitE supplementation was associated with attenuated FEV1 decline. The response to supplementation differed by rs2108622 such that individuals with the C allele, compared with the T allele, may need a higher dietary intake to reach the same plasma vitE concentration.

[The Effect of CYP4 F2 Polymorphism on Initial Warfarin Dose in Patients with Heart Valve Replacement].

OBJECTIVE: To study the effect of cytochrome P-4504F2 ( CYP4 F2) gene polymorphism on the initial dose of warfarin in patients after mechanical heart valve replacement. METHODS: We collected 350 patients receiving warfarin after mechanical heart valve replacement from January 2013 to December 2015 in our hospital. According to the international standardized ratio (INR) ≥2 at the initial stage after surgery, the patients were divided into two groups: INR≥2 group and INR<2 group. We selected the blood samples of all the 350 patients with testing the CYP4 F2 gene type of each patient, and analyzed the effect of CYP4 F2 gene polymorphism on the initial dose of warfarin after mechanical heart valve replacement (the average daily dose during hospitalization of patients 5-10 days after mechanical heart valve replacement). RESULTS: There was no statistical significance in the initial dose of warfarin among patients with different CYP4 F2 genotypes. However, warfarin dose was higher in CYP4 F2 TT genotype than in CYP4 F2 CC carriers ((3.37±0.68) mg vs. (2.94±0.74) mg, P<0.05) in INR≥2 group; In patients with the same genotype, the initial dose of warfarin in the CYP4 F2 CC ((4.02±0.58) mg vs. (2.94±0.74) mg) and CYP4 F2 CT genotypes ((4.15±0.88) mg vs. (3.18±0.82) mg) of INR<2 group was higher than that in INR≥2 group ( P<0.05). Gender, age, body mass index (BMI), comorbidities (hypertension, diabetes mellitus, coronary heart disease, atrial fibrillation), cytopigment P-450 2C9 ( CYP2 C9), CYP4 F2 and vitamin K peroxide-reductase complex 1 ( VKORC1) gene polymorphism and INR compliance were included in multiple linear regression analysis. The regression equation was as follows: warfarin initial dose (mg) =-8.634+0.352×BMI (kg/m 2) +1.102× CYP4 F2 genotype (CC or CT values 1, TT values 2) +2.147× VKORC1 (AA or AG values 1, GG values 2) +1.325×INR ( INR≥2 values 0, INR<2 values 1). The coefficient of determination ( R 2) of regression equation was 0.431 ( P<0.05). CONCLUSION: CYP4 F2 gene polymorphism may affect the initial dose of warfarin in patients after heart valve replacement, and this effect is also affected by body characteristics and other factors.

Influence of CYP4F2, ApoE, and CYP2A6 gene polymorphisms on the variability of Warfarin dosage requirements and susceptibility to cardiovascular disease in Jordan.

Cardiovascular diseases are among the leading causes of death worldwide. Many of those diseases require treatment with warfarin, an anticoagulant that has a large high inter and intra-variability in the required doses. The aim of this study is to find if there are any associations between rs2108622 of CYP4F2, rs7412 and rs405509 of ApoE, and rs1801272 of CYP2A6, and CVD and warfarin dose variability. The selected genes and their polymorphisms are involved in many GWAS associated with cardiovascular disease and variability in warfarin treatment. The study sample consisted of 212 Jordanian Cardiovascular patients and 213 healthy controls. DNA was extracted and the Mass ARRAY™ system was used to genotype four selected SNPs within three genes (CYP4F2, ApoE, and CYP2A6). Only one out of the four selected SNPs (ApoE rs7412 SNP) was found to be associated with the risk of cardiovascular disease. Also, this SNP showed significant differences in warfarin initial doses. CYP2A6 rs1801272 SNP was found to be associated with warfarin sensitivity during the initiation phase of therapy and with warfarin responsiveness and INR measurement during the stabilization phase of therapy. This study improves the current understanding of the high inter and intra-variabilities in response to warfarin, including the variety of dosing requirements and the susceptibility to cardiovascular disease in the Jordanian Arab population. Further study on a larger sample and in different ethnic groups could help in improving our understanding of warfarin's pharmacogenetics and its application in personalized medicine.

CYP4F2 and VKORC1 Polymorphisms Amplify the Risk of Carotid Plaque Formation.

INTRODUCTION: Atherosclerosis represents the process by which fibrous plaques are formed in the arterial wall, increasing its rigidity with a subsequent decrease in blood flow which can lead to several cardiovascular events. Seeing as vitamin K antagonists are involved in the pathogenesis of atherosclerosis, we decided to investigate whether polymorphisms in genes that influence vitamin K metabolism might have an impact in modulating the risk of plaque formation. PATIENTS AND METHODS: In the current study we included adult patients admitted in the Clinical Municipal Hospital of Cluj-Napoca without any carotid or femoral plaques clinically visible at the initial investigation, and a five year follow-up was subsequently performed. We recorded the following patient characteristics: age at inclusion, gender, area of living, smoking, presence of carotid and/or femoral plaques at five years, ischemic heart disease, arterial hypertension, atrial fibrillation, heart failure, diabetes mellitus, obesity, dyslipidemia, drug (oral anticoagulants, antihypertensives, hypolipidemic, anti-diabetic) use and status for the following gene polymorphisms: VKORC1 1639 G>A, CYP4F2 1347 G>T and GGCX 12970 C>G. RESULTS: We observed that the major predictor of both carotid and femoral plaque formation is represented by ischemic cardiac disease. VKORC1 and CYP4F2 polymorphisms did not predict plaque formation, except for VKORC1 homozygous mutants. Nonetheless, both VKORC1 and CYP4F2 interacted with ischemic cardiac disease, increasing the risk of developing a carotid plaque, while only CYP4F2, but not VKORC1, interacted with ischemic cardiac disease to increase the risk of femoral plaque formation. CONCLUSIONS: We documented that CYP4F2 and VKORC1 polymorphisms boost the proinflammatory plaque environment (observed indirectly through the presence of ischemic heart disease), increasing the risk of plaque development.

CYP4F2 and CYP3A5 gene polymorphisms and lung cancer in Chinese Han population.

This study aimed to explore whether the polymorphisms of CYP4F2 and CYP3A5 are correlated with the risk of lung cancer development. A case-control study was conducted among 510 patients with pathologically confirmed lung cancer as the case group and 504 healthy individuals as the control group. Four single-nucleotide polymorphisms of the CYP4F2 and CYP3A5 genes were genotyped, and their correlations with the risk of lung cancer were examined using Chi-square test and logistic regression analysis. Stratified analysis found that the rs3093105 and rs3093106 loci of CYP4F2 gene were significantly associated with lower risk of lung cancer (P = 0.012, OR 0.64, 95% CI 0.45-0.91). The correlation was related to patients' age and sex and pathological type of lung cancer. Similarly, the rs10242455 loci of CYP3A5 gene showed a statistical significance between the case group and the control group (P = 0.018, OR 0.71, 95% CI 0.53-0.94), which also was associated with reduced risk of squamous cell lung cancer in the dominant and additive models (dominant: OR 0.66, 95% CI 0.46-0.94, P = 0.021; additive: OR 0.71, 95% CI 0.53-0.95, P = 0.023). CYP4F2 and CYP3A5 gene polymorphisms are associated with the reduced risk of non-small cell lung cancer, and its correlation is related to patients' age and sex and pathological type of lung cancer.

The correlation between CYP4F2 variants and chronic obstructive pulmonary disease risk in Hainan Han population.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a complex pulmonary disease. Cytochrome P450 family 4 subfamily F member 2 (CYP4F2) belongs to cytochrome P450 superfamily of enzymes responsible for metabolism, its single nucleotide polymorphisms (SNPs) were reported to be involved in metabolism in the development of many diseases. The study aimed to assess the relation between CYP4F2 SNPs and COPD risk in the Hainan Han population. METHOD: We genotyped five SNPs in CYP4F2 in 313 cases and 508 controls by Agena MassARRAY assay. The association between CYP4F2 SNPs and COPD risk were assessed by χ2 test and genetic models. Besides, logistic regression analysis was introduced into the calculation for odds ratio (OR) and 95% confidence intervals (CIs). RESULTS: Allele model analysis indicated that rs3093203 A was significantly correlated with an increased risk of COPD. Also, rs3093193 G and rs3093110 G were associated with a reduced COPD risk. In the genetic models, we found that rs3093203 was related to an increased COPD risk, while rs3093193 and rs3093110 were related to a reduced risk of COPD. After gender stratification, rs3093203, rs3093193 and rs3093110 showed the association with COPD risk in males. With smoking stratification, rs3093144 was significantly associated with an increased risk of COPD in smokers. CYP4F2 SNPs were significantly associated with COPD risk. CONCLUSIONS: Our findings illustrated potential associations between CYP4F2 polymorphisms and COPD risk. However, large-scale and well-designed studies are needed to determine conclusively the association between the CYP4F2 SNPs and COPD risk.

Impact of gene polymorphism on the initiation and maintenance phases of warfarin therapy in Chinese patients undergoing heart valve replacement.

AIM: To determine whether VKORC1 rs9923231, CYP2C9 rs1057910, CYP4F2 rs2108622 and ORM1 rs17650 genotypes contribute to warfarin therapy in patients during initiation and maintenance anticoagulation treatment after heart valve surgery. METHODS: 287 Chinese patients with warfarin treatment more than three month after heart valve replacement operations were enrolled. Blood was collected from each subject for DNA extraction and genotyping. Analyzing the relationship between genotypes and warfarin curative effect. RESULTS: Their mean age was 48.0 ± 10.5 years old. During the initiation phase, the growth rate of INR was partial correlated with VKORC1 rs9923231, CYP2C9 rs1057910 and ORM1 rs17650, respectively. Compared with AG or GG genotypes of VKORC1 c.-1639 carriers, patients with VKORC1 c.-1639AA reached target INR therapeutic range faster (P<0.001) and has a high risk of overanticoagulation (P<0.001). Carriers of at least one CYP2C9 *3 allele reached the target INR therapeutic range and supra-therapeutic INR were faster than CYP2C9 wild-type carriers (P=0.032, P=0.032, respectively). CYP4F2 rs2108622 could significantly influence on time to the target INR therapeutic range and time to INR above 3.0 after hierarchical analysis with VKORC1, CYP2C9 and ORM1 (P=0.011, P=0.044, respectively). VKORC1 rs9923231, CYP2C9 rs1057910 and ORM1 rs17650 were significantly influence the %TTR in three months (P=0.031, P=0.008, P=0.001, respectively). During the maintenance phase, VKORC1 c.-1639AA carriers spent more time at supra-therapeutic INRs (P<0.001). CYP2C9 rs1057910, CYP4F2 rs2108622 and ORM1 rs17650 gene variants did not affect outcome parameters in maintenance phase. CONCLUSIONS: This study found that genetic factors could significantly affected on warfarin therapy in Chinese. Meanwhile, genetic variations play a more important role in the initial phase than did in maintenance phase of warfarin therapy.

Involvement of CYP4F2 in the Metabolism of a Novel Monophosphate Ester Prodrug of Gemcitabine and Its Interaction Potential In Vitro.

Compound-3 is an oral monophosphate prodrug of gemcitabine. Previous data showed that Compound-3 was more potent than gemcitabine and it was orally active in a tumor xenograft model. In the present study, the metabolism of Compound-3 was investigated in several well-known in vitro matrices. While relatively stable in human and rat plasma, Compound-3 demonstrated noticeable metabolism in liver and intestinal microsomes in the presence of NADPH and human hepatocytes. Compound-3 could also be hydrolyzed by alkaline phosphatase, leading to gemcitabine formation. Metabolite identification using accurate mass- and information-based scan techniques revealed that Compound-3 was subjected to sequential metabolism, forming alcohol, aldehyde and carboxylic acid metabolites, respectively. Results from reaction phenotyping studies indicated that cytochrome P450 4F2 (CYP4F2) was a key CYP isozyme involved in Compound-3 metabolism. Interaction assays suggested that CYP4F2 activity could be inhibited by Compound-3 or an antiparasitic prodrug pafuramidine. Because CYP4F2 is a key CYP isozyme involved in the metabolism of eicosanoids and therapeutic drugs, clinical relevance of drug-drug interactions mediated via CYP4F2 inhibition warrants further investigation.

Nonalcoholic fatty liver disease impairs the cytochrome P-450-dependent metabolism of α-tocopherol (vitamin E).

This study aims to investigate in in vivo and in vitro models of nonalcoholic fatty liver disease (NAFLD) the enzymatic metabolism of α-tocopherol (vitamin E) and its relationship to vitamin E-responsive genes with key role in the lipid metabolism and detoxification of the liver. The experimental models included mice fed a high-fat diet combined or not with fructose (HFD+F) and HepG2 human hepatocarcinoma cells treated with the lipogenic agents palmitate, oleate or fructose. CYP4F2 protein, a cytochrome P-450 isoform with proposed α-tocopherol ω-hydroxylase activity, decreased in HFD and even more in HFD+F mice liver; this finding was associated with increased hepatic levels of α-tocopherol and decreased formation of the corresponding long-chain metabolites α-13-hydroxy and α-13-carboxy chromanols. A decreased expression was also observed for PPAR-γ and SREBP-1 proteins, two vitamin E-responsive genes with key role in lipid metabolism and CYP4F2 gene regulation. A transient activation of CYP4F2 gene followed by a repression response was observed in HepG2 cells during the exposure to increasing levels of the lipogenic and cytotoxic agent palmitic acid; such gene repression effect was further exacerbated by the co-treatment with oleic acid and α-tocopherol and was also observed for PPAR-γ and the SREBP isoforms 1 and 2. Such gene response was associated with increased uptake and ω-hydroxylation of α-tocopherol, which suggests a minor role of CYP4F2 in the enzymatic metabolism of vitamin E in HepG2 cells. In conclusion, the liver metabolism and gene response of α-tocopherol are impaired in experimental NAFLD.

Association between CYP4F2 genotype and circulating plasma vitamin K concentration in children on chronic warfarin therapy: Possible long-term implications for bone development and vascular health.

Vitamin K is essential, for the activation of clotting proteins, as well as the biosynthesis of osteocalcin in bones and the activation of matrix-Gla protein needed in maintaining vasculature health. Cytochrome p450 4F2 (CYP4F2) enzyme is involved in vitamin K catabolism. Genetic polymorphism in CYP4F2 is thus likely to affect vitamin K systemic availability. We show that children on chronic warfarin therapy have low levels of vitamin K and vitamin K levels are linked to CYP4F2 genotype. Long-term low levels of vitamin K, influenced by CYP4F2 genotype, might affect bone development and vascular health in children on chronic warfarin therapy.

The impact of non-genetic and genetic factors on a stable warfarin dose in Thai patients.

PURPOSE: The aim of this study was to investigate the contributions of non-genetic and genetic factors on the variability of stable warfarin doses in Thai patients. METHODS: A total of 250 Thai patients with stable warfarin doses were enrolled in the study. Demographics and clinical data, e.g., age, body mass index, indications for warfarin and concomitant medications, were documented. Four single nucleotide polymorphisms in the VKORC1 - 1639G > A, CYP2C9*3, CYP4F2 rs2108622, and UGT1A1 rs887829 genes were detected from gDNA using TaqMan allelic discrimination assays. RESULTS: The patients with variant genotypes of VKORC1 - 1639G > A required significantly lower warfarin stable weekly doses (SWDs) than those with wild-type genotype (p < 0.001). Similarly, the patients with CYP2C9*3 variant allele required significantly lower warfarin SWDs than those with homozygous wild-type (p = 0.006). In contrast, there were no significant differences in the SWDs between the patients who carried variant alleles of CYP4F2 rs2108622 and UGT1A1 rs887829 as compared to wild-type allele carriers. Multivariate analysis, however, showed that CYP4F2 rs2108622 TT genotype accounted for a modest part of warfarin dose variability (1.2%). In contrast, VKORC1 - 1639G > A, CYP2C9*3, CYP4F2 rs2108622 genotypes and non-genetic factors accounted for 51.3% of dose variability. CONCLUSIONS: VKORC1 - 1639G > A, CYP2C9*3, and CYP4F2 rs2108622 polymorphisms together with age, body mass index, antiplatelet drug use, amiodarone use, and current smoker status explained 51.3% of individual variability in stable warfarin doses. In contrast, the UGT1A1 rs887829 polymorphism did not contribute to dose variability.

CYP4F2 repression and a modified alpha-tocopherol (vitamin E) metabolism are two independent consequences of ethanol toxicity in human hepatocytes.

The expression of CYP4F2, a form of cytochrome P-450 with proposed role in α-tocopherol and long-chain fatty acid metabolism, was explored in HepG2 and HepaRG human hepatocytes during ethanol toxicity. Cytotoxicity, ROS production, and JNK and ERK1/2 kinase signaling increased in a dose and time-dependent manner during ethanol treatments; CYP4F2 gene expression decreased, while other CYP4F forms, namely 4F11 and 12, increased along with 3A4 and 2E1 isoforms. α-Tocopherol antagonized the cytotoxicity and CYP4F2 gene repression effect of ethanol in HepG2 cells. Ethanol stimulated the tocopherol-ω-hydroxylase activity and the other steps of vitamin E metabolism, which points to a minor role of CYP4F2 in this metabolism of human hepatocytes. PPAR-γ and SREBP-1c followed the same expression pattern of CYP4F2 in response to ethanol and α-tocopherol treatments. Moreover, the pharmacological inhibition of PPAR-γ synergized with ethanol in decreasing CYP4F2 protein expression, which suggests a role of this nuclear receptor in CYP4F2 transcriptional regulation. In conclusion, ethanol toxicity modifies the CYP expression pattern of human hepatic cells impairing CYP4F2 transcription and protein expression. These changes were associated with a lowered expression of the fatty acid biosynthesis regulators PPAR-γ and SREBP-1c, and with an increased enzymatic catabolism of vitamin E. CYP4F2 gene repression and a sustained vitamin E metabolism appear to be independent effects of ethanol toxicity in human hepatocytes.

Human serum determination and in vitro anti-inflammatory activity of the vitamin E metabolite α-(13'-hydroxy)-6-hydroxychroman.

Cytochrome P450-derived long-chain metabolites are gaining increasing interest as bioactive intermediates of vitamin E. In this study we first report on the HPLC-ECD and GC-MS analysis in human serum of the earliest metabolite of this vitamin, namely α-(13'-hydroxy)-6-hydroxychroman (α-13'-OH). The two chromatographic procedure are sensitive enough (LOQ of 10nM) to measure α-13'-OH after hexane extraction of 1 ml of sample obtained from healthy volunteers supplemented for 1-week with 1000 IU/d (671 mg/d) RRR-α-tocopherol. The observed concentrations ranged between 15 and 50 nM, with minor differences between fasting and 4-hr post-meal state. Baseline (non-supplemented state) levels of 7.2 ± 1.6 nM were observed extracting higher volumes of serum. Biological effects of α-13'-OH investigated for the first time in RAW264.7 murine macrophages involved transcriptional control of inflammatory cytokines, and transcriptional and functional regulation of COX2 and iNOS enzymes in response to lipopolysaccharides. In conclusion, here we present the first quantitative evaluation of serum α-13'-OH also providing early evidence of the anti-inflammatory potential of this metabolite that is worth of further investigation in the area of functional and nutraceutical implications of vitamin E metabolism.

Intakes of omega-3 polyunsaturated fatty acids and blood pressure change over time: Possible interaction with genes involved in 20-HETE and EETs metabolism.

BACKGROUND: A high intake of omega-3 polyunsaturated fatty acids (ω-3 PUFAs), has been associated with reduced levels of blood pressure (BP). Their antihypertensive action may be due to the reduction of the ω-6/ω-3 ratio and the resulting competitive effect of ω-3 as compared to arachidonic acid (an ω-6 PUFA) as a substrate of cytochrome P450 (CYP450) enzymes involved in the production of vasoactive mediators. Some functional polymorphisms (SNPs), in genes which encode for the same enzymes, were associated with hypertension and ischemic stroke in the Malmö Diet and Cancer (MDC), a Swedish urban-based longitudinal study. The aim of this study was to evaluate the effect of the intake of different types of PUFAs on BP change over time (Δ-BP; mean follow-up 16.6±1.5 years; n=3.550 with complete phenotypic data), also considering the interaction with SNPs in genes involved in their metabolism via CYP450. METHODS: PUFA intakes were collected by a modified diet history method, and functional SNPs in CYP4F2, CYP4A11, CYP2J2 and EPHX2 were genotyped by Taqman. RESULT: We did not find any overall association between ω-6 and ω-3 PUFA intakes, or their ratio, with Δ-BP but observed an interaction between CYP4F2 V433M genotype and total omega-3, α-linolenic acid and linoleic/α-linolenic ratio, so that a higher ω-3 PUFA intake was significantly associated with a more pronounced BP decrease over time in subjects with the 433VV genotype (-0.041±0.018 mmHg/year; p=0.024; p-interaction=0.031) CONCLUSIONS: Our data do not support a major role of ω-6 or ω-3 PUFA intakes on BP change over time, but suggest a possible interaction of ω-3 PUFA with the CYP4F2 V433M.

Vascular characterization of mice with endothelial expression of cytochrome P450 4F2.

Cytochrome P450 (CYP) 4A and 4F enzymes metabolize arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). Although CYP4A-derived 20-HETE is known to have prohypertensive and proangiogenic properties, the effects of CYP4F-derived metabolites are not well characterized. To investigate the role of CYP4F2 in vascular disease, we generated mice with endothelial expression of human CYP4F2 (Tie2-CYP4F2-Tr). LC/MS/MS analysis revealed 2-foldincreases in 20-HETE levels in tissues and endothelial cells (ECs), relative to wild-type (WT) controls. Tie2-CYP4F2-Tr ECs demonstrated increases in growth (267.1 ± 33.4 vs. 205.0 ± 13% at 48 h) and tube formation (7.7 ± 1.1 vs. 1.6 ± 0.5 tubes/field) that were 20-HETE dependent and associated with up-regulation of prooxidant NADPH oxidase and proangiogenic VEGF. Increases in VEGF and NADPH oxidase levels were abrogated by inhibitors of NADPH oxidase and MAPK, respectively, suggesting the possibility of crosstalk between pathways. Interestingly, IL-6 levels in Tie2-CYP4F2-Tr mice (18.6 ± 2.7 vs. 7.9 ± 2.7 pg/ml) were up-regulated via NADPH oxidase- and 20-HETE-dependent mechanisms. Although Tie2-CYP4F2-Tr aortas displayed increased vasoconstriction, vasorelaxation and blood pressure were unchanged. Our findings indicate that human CYP4F2 significantly increases 20-HETE production, CYP4F2-derived 20-HETE mediates EC proliferation and angiogenesis via VEGF- and NADPH oxidase-dependent manners, and the Tie2-CYP4F2-Tr mouse is a novel model for examining the pathophysiological effects of CYP4F2-derived 20-HETE in the vasculature.-Cheng, J., Edin, M. L., Hoopes, S. L., Li, H., Bradbury, J. A., Graves, J. P., DeGraff, L. M., Lih, F. B., Garcia, V., Shaik, J. S. B., Tomer, K. B., Flake, G. P., Falck, J. R., Lee, C. R., Poloyac, S. M., Schwartzman, M. L., Zeldin, D. C. Vascular characterization of mice with endothelial expression of cytochrome P450 4F2.

ω-Hydroxylation of phylloquinone by CYP4F2 is not increased by α-tocopherol.

SCOPE: The objective of this study was to investigate the initial catabolic step of vitamin E and K metabolism, the ω-hydroxylation by human cytochrome P450 4F2 (CYP4F2). METHODS AND RESULTS: Tocopherol (T) metabolism was compared using rat liver slices incubated with deuterated (d6)-RRR-α-T (d6-α-T), racemic 2S-α-T (2S, 4'RS, 8'RS α-T, 2S-α-T), or d2-γ-T (d2-γ-T). Following comparable uptake of each T by liver slices, twice as much 13'-OH-T was produced from 2S-α-T or d2-γ-T (39 ± 15 or 42 ± 5 pmol/g liver, respectively) as from d6-α-T (17 ± 2, p < 0.01). Kinetic studies were conducted using insect microsomes expressing human CYP4F2 incubated with d4-phylloquinone (d4-PK), d6-RRR-α-T, d3-SRR-α-T, or d2-γ-T. CYP4F2 demonstrated similar apparent maximal velocities (Vmax) when either of the α-Ts were used as substrates, which were less than the apparent d4-PK Vmax (p < 0.0002), while the CYP4F2 catalytic efficiency toward d4-PK (15.8 Vmax/Km) was five times greater than for α-Ts. Vitamin K had no effect on vitamin E catabolism, while vitamin E slightly decreased the d4-PK Vmax. CONCLUSION: CYP4F2 discriminates between Ts and PK in vitro, but α-T does not apparently increase PK ω-hydroxylation by this mechanism.