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Purpose: Abnormalities in lipid metabolism have been proposed in Bietti's crystalline dystrophy (BCD). We aim to characterize the lipid profiles in a case-control study., Methods: All participants were genetically confirmed by CYP4V2 gene sequencing and underwent chorioretinopathy evaluation by calculating the percentages of AF atrophy (PAFA). Fasting blood samples of BCD patients and controls were collected, and plasma was analyzed for routine lipid profiles. Targeted lipidomic evaluation includes long chain polyunsaturated fatty acids (LCPUFA) and associated eicosanoid metabolites., Results: Routine lipids profiles showed elevated plasma levels of triglyceride (P = 0.043) and low-density lipoprotein cholesterol (P = 0.024) in BCD patients. Lipidomic analysis showed significantly decreased levels of ω-3 LCPUFA including docosahexaenoic acid (DHA, 22:6, P = 0.00068) and eicosapentaenoic acid (EPA, 20:5, P = 0.0016), as well as ω-6 LCPUFA arachidonic acid (ARA, 20:4, P < 0.0001) in BCD patients. Eicosanoid metabolites, either derived from ω-3 and/ or ω-6 LCPUFAs via cyclooxygenase (COX) or lipoxygenase (LOX) pathways, including 5-HEPE, 12-HEPE, 13-HDHA, 15-HETE, 12-HETE, 5-HETE, 6k-PGF1a, PGE2, PGJ2, and TXB2, exhibited significant differences (P < 0.0001) between BCD patients and controls. Genotypes of CYP4V2, specifically the biallelic null mutations, were observed to correlate with more remarkably reduced levels of oxylipins, involving major LOX pathway metabolites including 5-HETE, 5-HEPE, 12-HEPE and LTB4., Conclusions: BCD patients demonstrated significant decreases in plasma levels of ω-3 and ω-6 LCPUFA (DHA, EPA, and ARA), as well as their downstream metabolites via the COX and LOX pathways, suggesting that these might be implicated in BCD pathogenesis and could serve as biomarkers and therapeutic targets of the disease., Key Messages: What is known BCD is a vision-threatening hereditary disease the causative gene of which is CYP4V2. Abnormalities in lipid metabolism have been proposed and demonstrated previously in BCD studies. The detailed pathogenesis remains unclear and controversial. What is new We observed prominent lipidomic alterations in the circulation when compared with age, gender, and bodymass index (BMI)-matched healthy controls. BCD patients demonstrated significant decreases in plasma levels of ω-3 and ω-6 LCPUFA (DHA, EPA, and ARA). Remarkable changes were observed in the downstream metabolites of the LCPUFA via the COX and LOX pathways. Genotypes of CYP4V2, specifically the biallelic null mutations, were observed to correlate with more remarkably reduced levels of oxylipins, involving major LOX pathway metabolites., Competing Interests: Declarations. Ethical approval: Institutional Review Board (IRB) approval was obtained from the Ethics Committee of Beijing Tongren Hospital, Capital Medical University, Beijing, China, and the current study adhered to the tenets of the Declaration of Helsinki. Research involving human participants and/or animals: BCD patients and healthy controls. Informed consent: Was adopted from each participant. Conflicts of interest: None., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Fatty acid omega hydroxylase P450s consist of enzymes that hydroxylate various chain-length saturated and unsaturated fatty acids (FAs) and bioactive eicosanoid lipids. The human cytochrome P450 gene 4 family (CYP4) consists of 12 members that are associated with several human diseases. However, their role in the progression of metabolic dysfunction-associated fatty liver disease (MASLD) remains largely unknown. It has long been thought that the induction of CYP4 family P450 during fasting and starvation prevents FA-related lipotoxicity through FA metabolism to dicarboxylic acids that are chain-shortened in peroxisomes and then transported to the mitochondria for complete oxidation. Several studies have revealed that peroxisome succinate transported to the mitochondria is used for gluconeogenesis during fasting and starvation, and recent evidence suggests that peroxisome acetate can be utilized for lipogenesis and lipid droplet formation as well as epigenetic modification of gene transcription. In addition, omega hydroxylation of the bioactive eicosanoid arachidonic acid to 20-Hydroxyeicosatetraenoic acid (20-HETE) is essential for activating the GPR75 receptor, leading to vasoconstriction and cell proliferation. Several mouse models of diet-induced MASLD have revealed the induction of selective CYP4A members and the suppression of CYP4F during steatosis and steatohepatitis, suggesting a critical metabolic role in the progression of fatty liver disease. Thus, to further investigate the functional roles of CYP4 genes, we analyzed the differential gene expression of 12 members of CYP4 gene family in datasets from the Gene Expression Omnibus (GEO) from patients with steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. We also observed the differential expression of various CYP4 genes in the progression of MASLD, indicating that different CYP4 members may have unique functional roles in the metabolism of specific FAs and eicosanoids at various stages of fatty liver disease. These results suggest that targeting selective members of the CYP4A family is a viable therapeutic approach for treating and managing MASLD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Scutellaria baicalensis Georgi (Scutellariae Radix, SR) and Coptis chinensis Franch (Coptidis Rhizoma, CR) is a classic herbal pair used in many Traditional Chinese Medicine formulations in the treatment of hyperlipidemia (HLP). As effective ingredients of the drug pair, the effects and mechanisms of berberine and baicalin in the treatment of HLP in the form of components compatibility are still unclear., Aim of the Study: To explore the mechanism of the components compatibility of SR and CR in the treatment of HLP., Materials and Methods: The HLP model was established by a high-fat diet. Serum biochemical indexes were detected. Transcriptomics and metabolomics were detected. RT-PCR and Western Blot were used to analyze the effect of RA on the expression of the Cyp4a family during the treatment of HLP., Results: Berberine-baicalin (RA) has a good effect in the treatment of HLP. RA can significantly reduce the body weight and liver weight of HLP, reduce the levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C), and increase the level of high-density lipoprotein (HDL-C). Through transcriptomic analysis, RA significantly reversed the gene expression of Cyp4a10, Cyp4a12 b, Cyp4a31, and Cyp4a32 in cytochrome P450 family 4 subfamily a (Cyp4a) which related to fatty acid degradation in the liver of HLP mice. The results of fatty acid detection showed that RA could significantly regulate heptanoic acid, EPA, adrenic acid, DH-γ-linolenic acid, and DPA in the cecum of HLP mice. The Cyp4a family genes regulated by RA are closely related to a variety of fatty acids regulated by RA. RT-PCR confirmed that RA could regulate Cyp4a mRNA expression in HLP mice. WB also showed that RA can regulate the protein expression level of Cyp4a., Conclusion: The components compatibility of SR and CR can effectively improve the blood lipid level of HLP mice, its mechanism may be related to regulating Cyp4a gene expression and affecting fatty acid degradation, regulating the level of fatty acid metabolism in the body., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bietti crystalline dystrophy (BCD) is a rare heritable retinal disease characterized by crystal deposition primarily in the retina. It is associated with atrophy of the retinal pigment epithelium (RPE) and is caused by variants in CYP4V2 , which encodes a cytochrome P450 hemethiolate protein superfamily member. CYP4V2 is involved in the selective hydrolysis of saturated medium chain fatty acids, and patients with BCD demonstrate abnormalities in fatty acid metabolism, including abnormal lipid profiles and the accumulation of the pathogenic crystals within the RPE, which leads to the visual pathologies characteristic of BCD. However, the precise identity of the crystals is currently unknown, and BCD has no established extraocular manifestations. Here, we report granulomatous hepatitis associated with abundant diffuse crystalline clefts in the hepatic parenchyma in 3 patients with retinal dystrophy and dyslipidemia: 2 with pathogenic CYP4V2 variants and 1 patient with clinical ophthalmologic findings suggestive of BCD but without available genetic testing. The unique and striking histologic features unifying the liver biopsies in all 3 patients strongly support a process related to abnormal fatty acid metabolism underlying the genetic disease of BCD, expanding the spectrum of BCD and shedding light on the importance of CYP4V2 in systemic fatty acid metabolism., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Patient-specific induced pluripotent stem cell-derived (iPSC-derived) cell lines allow for therapies to be tailored to individual patients, increasing therapeutic precision and efficiency. Bietti crystalline dystrophy (BCD) is a rare blinding disease estimated to affect about 67,000 individuals worldwide. Here, we used iPSC-derived retinal pigment epithelium (iRPE) cells from patients with BCD to evaluate adeno-associated virus-mediated (AAV-mediated) gene augmentation therapy strategies. We found that BCD iRPE cells were vulnerable to blue light-induced oxidative stress and that cellular phenotype can be quantified using 3 robust biomarkers: reactive oxygen species (ROS), 4-hydroxy 2-nonenal (4-HNE) levels, and cell death rate. Additionally, we demonstrated that AAV-mediated gene therapy can significantly reduce light-induced cell death in BCD iRPE cells. This is the first proof-of-concept study to our knowledge to show that AAV-CYP4V2 gene therapy can be used to treat light-induced RPE damage in BCD. Furthermore, we observed significant variability in cellular phenotypes among iRPE from patients with BCD of divergent mutations, which outlined genotype-phenotype correlations in BCD patient-specific cell disease models. Our results reveal that patient-specific iRPE cells retained personalized responses to AAV-mediated gene therapy. Therefore, this approach can advance BCD therapy and set a precedent for precision medicine in other diseases, emphasizing the necessity for personalization in healthcare to accommodate individual diversity.

Current therapeutic strategies for esophageal cancer (EC) patients have yielded limited improvements in survival rates. Recent research has highlighted the influence of drug metabolism enzymes on both drug response and EC development. Our study aims to identify specific drug metabolism enzymes regulated by histone acetylation and to elucidate its molecular and clinical features. CYP4F12 exhibited a notable upregulation subsequent to trichostatin A treatment as evidenced by RNA sequencing analysis conducted on the KYSE-150 cell line. The change in gene expression was associated with increased acetylation level of histone 3 K18 and K27 in the promoter. The regulation was dependent on p300. In silicon analysis of both The Cancer Genome Atlas esophageal carcinoma and GSE53624 dataset suggested a critical role of CYP4F12 in EC development, because CYP4F12 was downregulated in tumor tissues and predicted better disease-free survival. Gene ontology analysis has uncovered a robust correlation between CYP4F12 and processes related to cell migration, as well as its involvement in cytosine-mediated immune activities. Further investigation into the relationship between immune cells and CYP4F12 expression has indicated an increased level of B cell infiltration in samples with high CYP4F12 expression. CYP4F12 was also negatively correlated with the expression of inhibitory checkpoints. An accurate predictive nomogram model was established combining with clinical factors and CYP4F12 expression. In conclusion, CYP4F12 was crucial in EC development, and targeting CYP4F12 may improve the therapeutic efficacy of current treatment in EC patients. SIGNIFICANCE STATEMENT: CYP4F12 expression was downregulated in esophageal cancer (EC) patients and could be induced by trichostatin A. During EC development, CYP4F12 was linked to reduced cell migration and increased infiltration of B cells. CYP4F12 also is a biomarker as prognostic predictors and therapeutic guide in EC patients., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

The activation of pregnane X receptor (PXR) or peroxisome proliferator-activated receptor α (PPARα) can induce liver enlargement. Recently, we reported that PXR or PPARα activation-induced hepatomegaly depends on yes-associated protein (YAP) signaling and is characterized by hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area. However, it remains unclear whether PXR or PPARα activation-induced hepatomegaly can be reversed after the withdrawal of their agonists. In this study, we investigated the regression of enlarged liver to normal size following the withdrawal of PCN or WY-14643 (typical agonists of mouse PXR or PPARα) in C57BL/6 mice. The immunohistochemistry analysis of CTNNB1 and KI67 showed a reversal of hepatocyte size and a decrease in hepatocyte proliferation after the withdrawal of agonists. In details, the expression of PXR or PPARα downstream proteins (CYP3A11, CYP2B10, ACOX1, and CYP4A) and the expression of proliferation-related proteins (CCNA1, CCND1, and PCNA) returned to the normal levels. Furthermore, YAP and its downstream proteins (CTGF, CYR61, and ANKRD1) also restored to the normal states, which was consistent with the change in liver size. These findings demonstrate the reversibility of PXR or PPARα activation-induced hepatomegaly and provide new data for the safety of PXR and PPARα as drug targets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Liver metastasis stands as the primary contributor to mortality among patients diagnosed with colorectal cancer (CRC). Neutrophil extracellular traps (NETs) emerge as pivotal players in the progression and metastasis of cancer, showcasing promise as prognostic biomarkers. Our objective is to formulate a predictive model grounded in genes associated with neutrophil extracellular traps and identify novel therapeutic targets for combating CRLM. We sourced gene expression profiles from the Gene Expression Omnibus (GEO) database. Neutrophil extracellular trap-related gene set was obtained from relevant literature and cross-referenced with the GEO datasets. Differentially expressed genes (DEGs) were identified through screening via the least absolute shrinkage and selection operator regression and random forest modeling, leading to the establishment of a nomogram and subtype analysis. Subsequently, a thorough analysis of the characteristic gene CYP4F3 was undertaken, and our findings were corroborated through immunohistochemical staining. We identified seven DEGs (ATG7, CTSG, CYP4F3, F3, IL1B, PDE4B, and TNF) and established nomograms for the occurrence and prognosis of CRLM. CYP4F3 is highly expressed in CRC and colorectal liver metastasis (CRLM), exhibiting a negative correlation with CRLM prognosis. It may serve as a potential therapeutic target for CRLM. A novel prognostic signature related to NETs has been developed, with CYP4F3 identified as a risk factor and potential target for CRLM., (© 2024. The Author(s).)

Cytochrome P450 (CYP)4Z1, a highly expressed CYP gene in breast cancer, was one of the last CYPs to be identified in the human genome, some 20 years ago. CYP4 enzymes typically catalyze ω -hydroxylation and metabolize ω 3 and ω 6 polyunsaturated fatty acids to bioactive lipid metabolites that can influence tumor growth and metastasis. These attributes of CYP4Z1 make it an attractive target for new chemotherapeutic drug design, as a potential biomarker for selection of patients that might respond favorably to drugs and for developing enzyme inhibitors as potential therapeutic agents. This review summarizes the current state of knowledge regarding the advancing biochemistry of CYP4Z1, its role in breast cancer, and the recent synthesis of selective chemical inhibitors of the enzyme. We identify gaps that need to be filled to further advance this field and present new experimental data on recombinant CYP4Z1 expression and purification of the active catalytic form. SIGNIFICANCE STATEMENT: In breast cancer, an unmet need is the availability of highly effective therapeutic agents, especially for triple negative breast cancer. The relevance of the work summarized in this mini-review is that it identifies a new potential drug target, CYP4Z1, and discusses ways in which the gene product's catalytic activity might be modulated in order to combat this malignancy and limit its spread., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease without approved therapeutic drugs. It is caused by mutations in CYP4V2 gene, and about 80% of BCD patients carry mutations in exon 7 to 11. Here, we apply CRISPR/Cas9 mediated homology-independent targeted integration (HITI)-based gene editing therapy in HEK293T cells, BCD patient derived iPSCs, and humanized Cyp4v3 mouse model (h-Cyp4v3 mut/mut ) using two rAAV2/8 vectors via sub-retinal administration. We find that sgRNA-guided Cas9 generates double-strand cleavage on intron 6 of the CYP4V2 gene, and the HITI donor inserts the carried sequence, part of intron 6, exon 7-11, and a stop codon into the DNA break, achieving precise integration, effective transcription and translation both in vitro and in vivo. HITI-based editing restores the viability of iPSC-RPE cells from BCD patient, improves the morphology, number and metabolism of RPE and photoreceptors in h-Cyp4v3 mut/mut mice. These results suggest that HITI-based editing could be a promising therapeutic strategy for those BCD patients carrying mutations in exon 7 to 11, and one injection will achieve lifelong effectiveness., (© 2024. The Author(s).)

Cytochrome P450 (CYP) 4Z1 (CYP4Z1) has recently garnered much interest as its expression predicts a poor prognosis and as a oncogene in breast cancer, and overexpressed in other many cancers. We previously showed that CYP4Z1 acts as a promoter of cancer stem cells (CSCs) to facilitate the occurrence and development of breast cancer. Here, RNA sequencing found that 1-benzylimidazole (1-Benzy) held a preferable correlation with breast cancer and suppressed the expression of CSC makers. Further functional experiments, including mammary spheroid formation, wound-healing, transwell-invasion, detection of tumor initiation, and metastatic ability, showed that 1-Benzy suppressed the stemness and metastasis of breast cancer cells. Additionally, we further demonstrated that CYP4Z1 is necessary for 1-Benzy-mediated suppression on breast cancer stemness and 1-Benzy exerted a weaker effect in breast cancer cells with CYP4Z1 knockdown. Taken together, our data suggest that 1-Benzy might be a potential drug suppressing breast cancer stemness via targeting CYP4Z1., (© 2023 Wiley Periodicals LLC.)

Cytochrome P450 4F2 (CYP4F2) is an enzyme that is involved in the metabolism of arachidonic acid (AA), vitamin E and K, and xenobiotics including drugs. CYP4F2*3 polymorphism (rs2108622; c.1297G>A; p.Val433Met) has been associated with hypertension, ischemic stroke, and variation in the effectiveness of the anticoagulant drug warfarin. In this study, we characterized wild-type CYP4F2 and 28 CYP4F2 variants, including a Val433Met substitution, detected in 8380 Japanese subjects. The CYP4F2 variants were heterologously expressed in 293FT cells to measure the concentrations of CYP4F2 variant holoenzymes using carbon monoxide-reduced difference spectroscopy, where the wild type and 18 holoenzyme variants showed a peak at 450 nm. Kinetic parameters [ V max , substrate concentration producing half of V max ( S 50 ), and intrinsic clearance (CL int ) as V max / S 50 ] of AA ω -hydroxylation were determined for the wild type and 21 variants with enzyme activity. Compared with the wild type, two variants showed significantly decreased CL int values for AA ω -hydroxylation. The values for seven variants could not be determined because no enzymatic activity was detected at the highest substrate concentration used. Three-dimensional structural modeling was performed to determine the reason for reduced enzymatic activity of the CYP4F2 variants. Our findings contribute to a better understanding of CYP4F2 variant-associated diseases and possible future therapeutic strategies. SIGNIFICANCE STATEMENT: CYP4F2 is involved in the metabolism of arachidonic acid and vitamin K, and CYP4F2*3 polymorphisms have been associated with hypertension and variation in the effectiveness of the anticoagulant drug warfarin. This study presents a functional analysis of 28 CYP4F2 variants identified in Japanese subjects, demonstrating that seven gene polymorphisms cause loss of CYP4F2 function, and proposes structural changes that lead to altered function., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

2-Alkylquinolones are a class of microbial natural products primarily produced in the Pseudomonas and Burkholderia genera that play a key role in modulating quorum sensing. Bacterial alkylquinolones were synthesized and then subjected to oxidative biotransformation using human cytochrome P450 enzyme CYP4F11, heterologously expressed in the fission yeast Schizosaccharomyces pombe . This yielded a range of hydroxylated and carboxylic acid derivatives which had undergone ω-oxidation of the 2-alkyl chain, the structures of which were determined by analysis of NMR and MS data. Oxidation efficiency depended on chain length, with a chain length of eight or nine carbon atoms proving optimal for high yields. Homology modeling suggested that Glu233 was relevant for binding, due to the formation of a hydrogen bond from the quinolone nitrogen to Glu233, and in this position only the longer alkyl chains could come close enough to the heme moiety for effective oxidation. In addition to the direct oxidation products, a number of esters were also isolated, which was attributed to the action of endogenous yeast enzymes on the newly formed ω-hydroxy-alkylquinolones. ω-Oxidation of the alkyl chain significantly reduced the antimicrobial and antibiofilm activity of the quinolones.

Malaria is a frequent parasitic infection becomes life threatening due to the disequilibrated immune responses of the host. Avid phagocytosis of malarial pigment hemozoin (HZ) and HZ-containing Plasmodium parasites incapacitates monocyte functions by bioactive lipoperoxidation products 4-hydroxynonenal (4-HNE) and hydroxyeicosatetraenoic acids (HETEs). CYP4F conjugation with 4-HNE is hypothesised to inhibit ω-hydroxylation of 15-HETE, leading to sustained monocyte dysfunction caused by 15-HETE accumulation. A combined immunochemical and mass-spectrometric approach identified 4-HNE-conjugated CYP4F11 in primary human HZ-laden and 4-HNE-treated monocytes. Six distinct 4-HNE-modified amino acid residues were revealed, of which C260 and H261 are localized in the substrate recognition site of CYP4F11. Functional consequences of enzyme modification were investigated on purified human CYP4F11. Palmitic acid, arachidonic acid, 12-HETE, and 15-HETE bound to unconjugated CYP4F11 with apparent dissociation constants of 52, 98, 38, and 73 µM, respectively, while in vitro conjugation with 4-HNE completely blocked substrate binding and enzymatic activity of CYP4F11. Gas chromatographic product profiles confirmed that unmodified CYP4F11 catalysed the ω-hydroxylation while 4-HNE-conjugated CYP4F11 did not. The 15-HETE dose dependently recapitulated the inhibition of the oxidative burst and dendritic cell differentiation by HZ. The inhibition of CYP4F11 by 4-HNE with consequent accumulation of 15-HETE is supposed to be a crucial step in immune suppression in monocytes and immune imbalance in malaria.

N-acyl amino acids are a large family of circulating lipid metabolites that modulate energy expenditure and fat mass in rodents. However, little is known about the regulation and potential cardiometabolic functions of N-acyl amino acids in humans. Here, we analyze the cardiometabolic phenotype associations and genomic associations of four plasma N-acyl amino acids (N-oleoyl-leucine, N-oleoyl-phenylalanine, N-oleoyl-serine, and N-oleoyl-glycine) in 2351 individuals from the Jackson Heart Study. We find that plasma levels of specific N-acyl amino acids are associated with cardiometabolic disease endpoints independent of free amino acid plasma levels and in patterns according to the amino acid head group. By integrating whole genome sequencing data with N-acyl amino acid levels, we identify that the genetic determinants of N-acyl amino acid levels also cluster according to the amino acid head group. Furthermore, we identify the CYP4F2 locus as a genetic determinant of plasma N-oleoyl-leucine and N-oleoyl-phenylalanine levels in human plasma. In experimental studies, we demonstrate that CYP4F2-mediated hydroxylation of N-oleoyl-leucine and N-oleoyl-phenylalanine results in metabolic diversification and production of many previously unknown lipid metabolites with varying characteristics of the fatty acid tail group, including several that structurally resemble fatty acid hydroxy fatty acids. These studies provide a structural framework for understanding the regulation and disease associations of N-acyl amino acids in humans and identify that the diversity of this lipid signaling family can be significantly expanded through CYP4F-mediated ω-hydroxylation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The cytochrome P450 (CYP)4 family of enzymes are monooxygenases responsible for the ω-oxidation of endogenous fatty acids and eicosanoids and play a crucial part in regulating numerous eicosanoid signaling pathways. Recently, CYP4 gained attention as a potential therapeutic target for several human diseases, including cancer, cardiovascular diseases and inflammation. Small-molecule inhibitors of CYP4 could provide promising treatments for these diseases. The aim of the present review is to highlight the advances in the field of CYP4, discussing the physiology and pathology of the CYP4 family and compiling CYP4 inhibitors into groups based on their chemical classes to provide clues for the future discovery of drug candidates targeting CYP4., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

α-Tocopherol (α-TOH) is a potent antioxidant. The concentrations of α-TOH in plasma are closely related to human health. α-TOH can be regulated by the metabolism of cytochrome P450 4F2 (CYP4F2). However, the atomic-level basis for this regulation process remains elusive. Here, we successfully constructed the structure of CYP4F2 by homology modeling and obtained the α-TOH-CYP4F2 complex models using molecular docking. Three parallel 500 ns molecular dynamics simulations were performed on each complex model to investigate the details of the interaction between α-TOH and CYP4F2. MM-GBSA method combined with principal component analysis shows that 8 key residues establish a hydrophobic cavity stabilizing α-TOH in the pocket of CYP4F2 and S423 forms an important hydrogen bond with α-TOH anchoring α-TOH in the favorable position for ω-hydroxylation. Based on our simulation results and the experimental facts, we designed mutation simulation experiments to clarify the important role of two key residues (S423 and V433) in the binding of α-TOH with CYP4F2. The results show that the mutations directly or indirectly change the binding mode of α-TOH and decrease its binding affinity with CYP4F2, which is unfavorable for ω-hydroxylation. Our results could enrich the information on structure-function relationships of CYP4F2 and provide valuable insights into the regulatory mechanism of CYP4F2 on the metabolism of α-TOH., (© 2023 Wiley Periodicals LLC.)

300 million people live with at least one of 6,000 rare diseases worldwide. However, rare disease research is not always reviewed with scrutiny, making it susceptible to what the author refers to as nontransparent science. Nontransparent science can obscure animal model flaws, misguide medicine regulators and drug developers, delay or frustrate orphan drug development, or waste limited resources for rare disease research. Flawed animal models not only lack pharmacologic relevance, but also give rise to issue of clinical translatability. Sadly, these consequences and risks are grossly overlooked. Nontransparency in science can take many forms, such as premature publication of animal models without clinically significant data, not providing corrections when flaws to the model are discovered, lack of warning of critical study limitations, missing critical control data, questionable data quality, surprising results without a sound explanation, failure to rule out potential factors which may affect study conclusions, lack of sufficient detail for others to replicate the study, dubious authorship and study accountability. Science has no boarders, neither does nontransparent science. Nontransparent science can happen irrespective of the researcher's senority, institutional affiliation or country. As a patient-turned researcher suffering from Bietti crystalline dystrophy (BCD), I use BCD as an example to analyze various forms of nontransparent science in rare disease research. This article analyzes three papers published by different research groups on Cyp4v3 -/- , high-fat diet (HFD)-Cyp4v3 -/- , and Exon1-Cyp4v3 -/- mouse models of BCD. As the discussion probes various forms of nontransparent science, the flaws of these knockout mouse models are uncovered. These mouse models do not mimic BCD in humans nor do they address the lack of Cyp4v3 (murine ortholog of human CYP4V2) expression in wild type (WT) mouse retina which is markedly different from CYP4V2 expression in human retina. Further, this article discusses the impact of nontransparent science on drug development which can lead to significant delays ultimately affecting the patients. Lessons from BCD research can be helpful to all those suffering from rare diseases. As a patient, I call for transparent science in rare disease research., (© 2023. The Author(s).)

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive retinal degenerative disease characterized by yellow-white crystal deposits in the posterior pole, degeneration of the retinal pigment epithelium (RPE), and sclerosis of the choroid. Mutations in the cytochrome P450 4V2 gene (CYP4V2) cause BCD, which is associated with lipid metabolic disruption. The use of gene-replacement therapy in BCD has been hampered by the lack of disease models. To advance CYP4V2 gene-replacement therapy, we generated BCD patient-specific induced pluripotent stem cell (iPSC)-RPE cells and Cyp4v3 knockout (KO) mice as disease models and AAV2/8-CAG-CYP4V2 as treatment vectors. We demonstrated that after adeno-associated virus (AAV)-mediated CYP4V2 gene-replacement therapy BCD-iPSC-RPE cells presented restored cell survival and reduced lipid droplets accumulation; restoration of vision in Cyp4v3 KO mice was revealed by elevated electroretinogram amplitude and ameliorated RPE degeneration. These results suggest that AAV-mediated gene-replacement therapy in BCD patients is a promising strategy., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The secondary metabolite pseudopyronine B, isolated from Pseudomonas mosselii P33, was biotransformed by human P450 enzymes, heterologously expressed in the fission yeast Schizosaccharomyces pombe . Small-scale studies confirmed that both CYP4F2 and CYP4F3A were capable of oxidizing the substrate, with the former achieving a higher yield. In larger-scale studies using CYP4F2, three new oxidation products were obtained, the structures of which were elucidated by UV-vis, 1D and 2D NMR, and HR-MS spectroscopy. These corresponded to hydroxylated, carboxylated, and ester derivatives ( 1 - 3 ) of pseudopyronine B, all of which had been oxidized exclusively at the ω-position of the C-6 alkyl chain. In silico homology modeling experiments highlighted key interactions between oxygen atoms of the pyrone ring and two serine residues and a histidine residue of CYP4F2, which hold the substrate in a suitable orientation for oxidation at the terminus of the C-6 alkyl chain. Additional modeling studies with all three pseudopyronines revealed that the seven-carbon alkyl chain of pseudopyronine B was the perfect length for oxidation, with the terminal carbon lying close to the heme iron. The antibacterial activity of the substrates and three oxidation products was also assessed, revealing that oxidation at the ω-position removes all antimicrobial activity. This study both increases the range of known substrates for human CYF4F2 and CYP4F3A enzymes and demonstrates their utility in producing additional natural product derivatives.

Significance: The identification of a role for CYP4F2-dependent metabolism in driving immune evasion in non-small cell lung cancer reveals a strategy to improve the efficacy of immunotherapy by inhibiting CYP4F2. See related article by Van Ginderachter, p. 3882., (©2022 American Association for Cancer Research.)

Background: Lymph node metastasis is an important route of lung cancer metastasis and can significantly affect the survival of lung cancer., Methods: All the analysis was conducted out in the R software. Expression profile and clinical information of lung adenocarcinoma (LUAD) patients were downloaded from The Cancer Genome Atlas database., Results: In our study, we firstly identified the characteristic genes of lymph node metastasis in LUAD through two machine learning algorithms, least absolute shrinkage and selection operator (LASSO) logistic regression, and SVM-RFE algorithms. Ten characteristic genes were finally identified, including CRHR2 , ITIH1 , PRSS48 , MAS1L , CYP4Z1 , LMO1 , TCP10L2 , KRT78 , IGFBP1 , and PITX3 . Next, we performed univariate Cox regression, LASSO regression, and multivariate Cox regression sequentially to construct a prognosis model based on MAS1L , TCP10L2 , and CRHR2 , which had a good prognosis prediction efficiency in both training and validation cohorts. Univariate and multivariate analysis indicated that our model is a risk factor independent of other clinical features. Pathway enrichment analysis showed that in the high-risk patients, the pathway of MYC target, unfolded protein response, interferon alpha response, DNA repair, reactive oxygen species pathway, and glycolysis were significantly enriched. Among three model genes, MAS1L aroused our interest and therefore was selected for further analysis. KM survival curves showed that the patients with higher MAS1L might have better disease-free survival and progression-free survival. Further, pathway enrichment, genomic instability, immune infiltration, and drug sensitivity analysis were performed to in-deep explore the role of MAS1L in LUAD., Conclusions: Results showed that the signature based on MAS1L , TCP10L2 , and CRHR2 is a useful tool to predict prognosis and lung cancer lymph node metastasis., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Qian Zhou et al.)

Background and Objective: Ovarian cancer is a leading cause of death in females. Since its treatment is challenging and causes severe side effects, novel therapies are urgently needed. One of the potential enzymes implicated in the progression of cancers is Cytochrome 4Z1 (CYP4Z1). Its expression in ovarian cancer remains unknown. Therefore, the current study aims to assess CYP4Z1 expression in different subtypes of ovarian cancers. Materials and Methods: Immunohistochemistry was used to characterize CYP4Z1 expression in 192 cases of ovarian cancers along with eight normal ovarian tissues. The enzyme’s association with various clinicopathological characteristics and survival was determined. Results: CYP4Z1 was strongly expressed in 79% of ovarian cancers, compared to negative expression in normal ovarian samples. Importantly, significantly high CYP4Z1 expres-sion was determined in patients with advanced-stage cancer and a high depth of invasion (p < 0.05). Surprisingly, CYP4Z1 expression was significantly associated with a low patient survival rate. Univariate analysis revealed that patient survival was strongly associated with CYP4Z1 expression, tumor stage, depth of invasion, and lymph node metastasis (p < 0.05). Multivariate analysis showed that only CYP4Z1 expression was significantly associated with patient survival (p < 0.05). Conclusions: CYP4Z1 expression is correlated with shorter patient survival and has been identified as an independent indicator of a poor prognosis for ovarian cancer patients.

Background: Insect cytochrome P450 monooxygenases (P450s) play a crucial role in phytochemical metabolism and tolerance. Three P450 genes (TcCYP4Q3, TcCYP4Q5, and TcCYP4Q7) are associated with the response of eugenol in Tribolium castaneum. However, the responding mechanisms of these P450 genes to eugenol remain unknown., Results: Here, spatiotemporal expression profiling revealed that TcCYP4Q3 and TcCYP4Q5 were most highly expressed in late adult, while TcCYP4Q7 was predominantly expressed in late larva; and all of these three P450 genes were mainly expressed in the fat body of larvae. Furthermore, the expressions of these three P450 genes were significantly up-regulated after exposure to eugenol, and depletion of them enhanced the susceptibility of beetles to eugenol. Interestingly, RNA interference (RNAi) against the CncC gene, a transcription factor of CncC signaling pathway associated with regulation of insect P450s in response to phytochemicals, reduced the transcripts of these three P450 genes following exposure to eugenol. Investigation of CncC signaling pathway showed that this pathway could be activated by eugenol., Conclusion: Altogether, the results indicate that these three P450 genes are regulated by CncC signaling pathway to participate in the susceptibility of Tribolium castaneum to phytochemicals. These findings will aid implications for the development of novel therapeutics to control pest. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Purpose: To investigate the relationship between different CYP4V2 disease-causing variants and disease severity in Bietti crystalline dystrophy (BCD)., Methods: Twenty-one subjects from 19 unrelated families with a clinical diagnosis of BCD were enrolled. A novel severity prediction score for BCD based on the predicted molecular impact of CYP4V2 variants was applied for grouping and subsequent analyses. The more severe variants led to less CYP4V2 protein function preservation and a higher severity prediction score., Results: All subjects harbored two alleles of CYP4V2 disease-causing variants, of which c.802-8&#95;810del17insGC was the most prevalent (14/21, 66.67%) and c.1507G>C was novel. According to the severity score, the subjects were categorized into severe, moderate, and mild groups with different preservation of central vision (mean logMAR visual acuity 0.95 ± 0.82, 0.89 ± 1.22, and 0.56 ± 0.64, respectively). The patients with a lower severity score had slower disease progression., Conclusion: This is the first cohort study of BCD in Taiwan, and we established a novel BCD severity index based on the molecular impact of different CYP4V2 variants. More severe impairment of CYP4V2 protein led to a more severe disease course with earlier progression. Our results could be helpful in identifying a therapeutic window for patients with BCD.

Objective: To investigate CYP4V2 gene variants and ocular clinical characteristics of Bietti corneoretinal crystalline dystrophy in China so as to provide more references for genotype and phenotype of BCD., Methods: Sixteen Chinese probands were recruited in Beijing Tongren Hospital in a retrospective study. All patients underwent CYP4V2 gene detection and ophthalmic clinical examinations., Results: CYP4V2 gene variants were detected in all patients. Eight variants were identified, and the most common one was c.802-8&#95;810del17bpinsGC. Onset age of BCD was from 12 to 44 years, and the first symptoms mostly were decreased visual acuity or night blindness. Corneal crystalline depositions were observed in all patients and were found not only in epithelium and superficial stroma near the limbus but also in corneal endothelium. OCT showed atrophy of RPE in all patients, outer retinal tubulation in ten patients, macular edema in four patients, macular hole in three patients with one accompanied with retinal detachment, and choroidal neovascularization in one patient., Conclusion: CYP4V2 gene variants were detected in all patients consistent with the genetic locus homogeneity of BCD, and c.802-8&#95;810del17bpinsGC was the most common mutation. Corneal crystalline depositions were observed in all patients, which may be features of BCD and helpful for the diagnosis of BCD patients, especially those in the advanced stage without typical fundus crystalline depositions or without gene detection. However, considerable phenotypic variability was detected. Corneal crystalline deposits were observed not only in epithelium and superficial stroma but also in endothelium, which has not been reported before. This may provide further evidence for the variable phenotypic expression between affected individuals.

Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of β-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1 -/- mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1 -/- liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1 -/- mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1 -/- mice in better understanding disease mechanism in fatty acid α-oxidation disorders.

Fatty acids are essential in maintaining cellular homeostasis by providing lipids for energy production, cell membrane integrity, protein modification, and the structural demands of proliferating cells. Fatty acids and their derivatives are critical bioactive signaling molecules that influence many cellular processes, including metabolism, cell survival, proliferation, migration, angiogenesis, and cell barrier function. The CYP4 Omega hydroxylase gene family hydroxylate various short, medium, long, and very-long-chain saturated, unsaturated and polyunsaturated fatty acids. Selective members of the CYP4 family metabolize vitamins and biochemicals with long alkyl side chains and bioactive prostaglandins, leukotrienes, and arachidonic acids. It is uncertain of the physiological role of different members of the CYP4 omega hydroxylase gene family in the metabolic control of physiological and pathological processes in the liver. CYP4V2 is a unique member of the CYP4 family. CYP4V2 inactivation in retinal pigment epithelial cells leads to cholesterol accumulation and Bietti's Crystalline Dystrophy (BCD) pathogenesis. This commentary provides information on the role CYP4V2 has in metabolic syndrome and nonalcoholic fatty liver disease progression. This is accomplished by identifying its role in BCD, its control of cholesterol synthesis and lipid droplet formation in C. elegans, and the putative function in cardiovascular disease and gastrointestinal/hepatic pathologies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: CYP4Z1 is a human cytochrome P450 enzyme involved in breast cancer progression and prognosis, but its functional role in these processes is not understood. In order to gain more insight into CYP4Z1's properties it was recombinantly expressed in a host animal that does not have an endogenous homologue., Methods: We generated a transgenic mouse model that specifically expresses human CYP4Z1 in breast tissue under the control of the whey acidic protein promoter. Complementary experiments were done using cell lines derived from human breast cell., Results: Induction of CYP4Z1 expression led to reduction of body weight, activity, and birth rates. Histological analysis revealed no evidence for tumor formation. However, a strong increase in estrogen receptor alpha was observed by immunohistochemistry; weaker but significantly increased immunoreactivity was also detected for collagen I and fibronectin. Overexpression of CYP4Z1 in the human breast cancer cell line MCF7 also led to increased ERα expression. Moreover, increased expression of both CYP4Z1 and ERα was observed in MCF-10A normal breast cells upon cocultivation with MCF-7 cells (with or without overexpression of CYP4Z1)., Conclusion: These data suggest that CYP4Z1 facilitates breast cancer development by induction of ERα expression via an as yet undefined mechanism., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Warfarin is the most commonly evaluated drug in pharmacogenetic-guided dosing studies. However, gaps remain regarding the influence of the genetic polymorphisms of CYP2C9, VKORC1, and CYP4F2 on specific pharmacodynamic parameters like the warfarin sensitivity index (WSI), prothrombin time international normalized ratio (PT-INR), and log-INR variability., Methods: A cross-sectional study was conducted in non-smoking adults receiving warfarin for at least 6 months. Their demographics, diagnoses, warfarin dosing regimen, concomitant drugs, PT-INR, and bleeding episodes were obtained. CYP2C9 (rs1057910-*3 and rs1799853-*2 alleles), CYP4F2 (rs2108622), and VKORC1 (rs9923231) polymorphisms were assessed using real-time polymerase chain reaction. Three genotype groups (I-III) were defined based on the combined genetic polymorphisms of CYP2C9 and VKORC1 from the FDA's recommendations. Key outcome measures included anticoagulation control, time spent in therapeutic range, stable warfarin dose, WSI, log-INR variability, and Warfarin Composite Measure (WCM)., Results: The study recruited 236 patients; 75 (31.8%) carried a functional CYP2C9 variant allele, and, 143 (60.6%) had at least one T allele in CYP4F2 and 133 (56.4%) had at least one T allele in VKORC1. Groups' II and III CYP2C9 and VKORC1 genotypes were observed with reduced stable warfarin dose, increased WSI, higher log-INR variability, and increased bleeding risk. The presence of *2 or *3 allele in CYP2C9 was observed with reduced stable warfarin doses akin to the presence of T alleles in VKORC1; however, the doses increased with T alleles in CYP4F2., Conclusion: The evaluated genetic polymorphisms significantly influenced all the pharmacodynamic parameters of warfarin. Evaluating CYP2C9, VKORC1, and CYP4F2 genetic polymorphisms prior to warfarin initiation is likely to optimize therapeutic response., (© 2021. Maj Institute of Pharmacology Polish Academy of Sciences.)

Omecamtiv mecarbil (OM) is a novel cardiac myosin activator that is currently in clinical development for the treatment of heart failure. The absorption and disposition of [ 14 C]OM (60 µCi) were studied after a single intravenous infusion (35 mg over 1 hour) or oral solution dose (35 mg) in 14 healthy male subjects. Mean recovery of the administered [ 14 C]OM dose was 85.1% and 86.5% over 336 hours for the intravenous and oral routes, respectively. After intravenous dosing, 47.8% and 37.3% of the dose was recovered in urine and feces, respectively; after oral dosing, 48.6% and 38.0% was recovered in urine and feces, respectively. Unchanged OM accounted for a minor percentage of radioactivity in urine (mean 7.7% of dose) and feces (mean 4.1% of dose) across all subjects. The major metabolites recovered in urine and feces were M3 (decarbamoylation product) and sequential metabolite M4 (lactam of M3), which accounted for means of 26.5% and 11.6% of the administered dose, respectively. The CYP4 family of enzymes was primarily responsible for the formation of M3 based on in vitro studies. Other metabolic pathways accounted for 14.9% of the administered dose. In pooled plasma, OM, M3, and M4 accounted for 83.8%, 6.0%, and 3.3% of the total [ 14 C]OM-related materials. No other plasma metabolites constituted more than 3% of the administered dose. The bioavailability for OM solution was 93.5% after rapid and extensive absorption. SIGNIFICANCE STATEMENT: This study characterized the absorption and disposition of OM, a novel small molecule being developed for the treatment of heart failure. OM was primarily cleared through metabolism by the CYP4 family through oxidative cleavage of a terminal carbamate moiety that resembles hydrolysis., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

8-[(1H-1,2,3-benzotriazol-1-yl)amino]octanoic acid (8-BOA) was recently identified as a selective and potent mechanism-based inactivator (MBI) of breast cancer-associated CYP4Z1 and exhibited favourable inhibitory activity in vitro, thus meriting in vivo characterization.The pharmacokinetics and metabolism of 8-BOA in rats was examined after a single IV bolus dose of 10 mg/kg. A biphasic time-concentration profile resulted in relatively low clearance and a prolonged elimination half-life.The major circulating metabolites identified in plasma were products of β -oxidation; congeners losing two and four methylene groups accounted for >50% of metabolites by peak area. The -(CH 2 ) 2 product was characterized previously as a CYP4Z1 MBI and so represents an active metabolite that may contribute to the desired pharmacological effect. Ex vivo analysis of total CYP content in rat liver and kidney microsomes showed that off-target CYP inactivation was minimal; liver microsomal probe substrate metabolism also demonstrated low off-target inactivation. Standard clinical chemistries provided no indication of acute toxicity. In silico simulations using the free concentration of 8-BOA in plasma suggested that the in vivo dose used here may effectively inactivate CYP4Z1 in a xenografted tumour.

Human cytochrome P450 enzyme CYP4Z1 represents a promising target for the treatment of a multitude of malignancies including breast cancer. The most active known non-covalent inhibitor (1-benzylimidazole) only shows low micromolar affinity to CYP4Z1. We report a new, highly active inhibitor for CYP4Z1 showing confirmed binding in an enzymatic assay and an IC 50 value of 63 ± 19 nM in stably transfected MCF-7 cells overexpressing CYP4Z1. The new inhibitor was identified by a systematically developed virtual screening protocol. Binding was rationalized using a carefully elaborated 3D pharmacophore hypothesis and thoroughly characterized using extensive molecular dynamics simulations and dynamic 3D pharmacophore (dynophore) analyses. This novel inhibitor represents a valuable pharmacological tool to accelerate characterization of the still understudied CYP4Z1 and might pave the way for a new treatment strategy in CYP4Z1-associated malignancies. The presented in silico model for predicting CYP4Z1 interaction provides novel mechanistic insights and revealed that the drug ozagrel interacts with CYP4Z1., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

The brown planthopper, Nilaparvata lugens, is one of the main insect pests of rice. The N. lugens gene NlCYP4CE1 encodes cytochrome P450 monooxygenase (P450), which is a key enzyme in the metabolism of the insecticide imidacloprid. Previous research has suggested that the expression of NlCYP4CE1 is induced by imidacloprid stress, but the effect of bacterial symbionts on its expression has not been determined. The results of this study show that exposure to subtoxic imidacloprid changed the structure of the bacterial symbiont community in N. lugens. Specifically, the total bacterial content increased, but the bacterial species diversity significantly decreased. Wolbachia accounted for the largest proportion of bacteria in N. lugens; its abundance significantly increased after subtoxic imidacloprid exposure. The transcript level of NlCYP4CE1 was significantly increased by imidacloprid, but this effect was significantly weakened after Wolbachia was cleared with tetracycline. This result suggests that Wolbachia enhances the expression of NlCYP4CE1 to promote the detoxification metabolic response to imidacloprid stress. Understanding the effect of bacterial symbionts on gene expression in the host provides a new perspective on interactions between insecticides and their target insect pests, and highlights that subtoxic imidacloprid exposure may raise the risk of insecticide resistance by altering the structure of bacterial symbiont communities., (© 2020 Institute of Zoology, Chinese Academy of Sciences.)

In this article, we found that 20-Hydroxyeicosatetraenoic acid (20-HETE) reduced Na/K-ATPase α1 expression via the ubiquitin-proteasome pathway. The ubiquitination level of Na/K-ATPase α1 protein was increased in 20-HETE-treated mouse cortical collecting duct cells and the kidney tissues of CYP4F2 transgenic mice. We also demonstrated that 20-HETE-induced high level phosphorylation of Na/K-ATPase α1 was necessary for its ubiquitination.The protein kinase C inhibitor sotrastaurin significantly reduced the phosphorylation of Na/K-ATPase α1 and increased the expression of Na/K-ATPase α1 although 20-HETE stimulus being applied at the same time. Moreover, high level of 20-HETE increased the expression and neddylation of Cullin3,which is an important ubiquitin E3 ligase in kidney. MLN4924, an inhibitor of NEDD8-activating enzyme, inhibited neddylation of Cullin3 and reversed the reduction of Na/K-ATPase α1 expression caused by 20-HETE. Thus, 20-HETE downregulates Na/K-ATPase α1 via the ubiquitination pathway, and phosphorylation of Na/K-ATPase α1 is a prerequisite to ubiquitination. Additionally, 20-HETE regulates Cullin3 expression via neddylation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Oxidative damage by reactive oxygen species (ROS) is one of the main contributors to cell injury and tissue damage in thalassemia patients. Recent studies suggest that ROS generation in non-transfusion-dependent (NTDT) patients occurs as a result of iron overload. Among the different sources of ROS, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes and cytochrome P450 (CYP450) have been proposed to be major contributors for oxidative stress in several diseases. However, the sources of ROS in patients with NTDT remain poorly understood. In this study, Hbb th3/+ mice, a mouse model for β-thalassemia, were used. These mice exhibit an unchanged or decreased expression of the major NOX isoforms, NOX1, NOX2 and NOX4, when compared to their C57BL/6 control littermates. However, a significant increase in the protein synthesis of CYP4A and CYP4F was observed in the Hbb th3/+ mice when compared to the C57BL/6 control mice. These changes were paralleled by an increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a CYP4A and CYP4F metabolite. Furthermore, these changes corroborate with onset of ROS production concomitant with liver injury. To our knowledge, this is the first report indicating that CYP450 4A and 4F-induced 20-HETE production mediates reactive oxygen species overgeneration in Hbb th3/+ mice through an NADPH-dependent pathway.

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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Introduction: 20-Hydroxyeicosatetraenoic acid (20-HETE) is the metabolite of cytochrome P450, which modulates blood pressure by inhibiting renal sodium transport. However, the molecular mechanisms underlying the role of 20-HETE in the development of obesity-related hypertension remain unclear, necessitating this study., Methods: Cytochrome P450 4F2 (CYP4F2) transgenic mice fed high-fat diet (HFD) were used as research animal models. The expression of renal ion transport molecules targeted by 20-HETE was evaluated by real-time PCR and Western blot (WB). The regulatory effect of 20-HETE and HFD on renal Na+-K+-2Cl- cotransporter, isoform 2 (NKCC2) was explored by immunoprecipitation, WB, and luciferase assay., Results: A 2-week HFD feeding dramatically decreased protein abundance but increased renal NKCC2 mRNA expression in CYP4F2 transgenic mice. The decrease in NKCC2 protein was demonstrated to be due to ubiquitination induced by the synergy between 20-HETE and HFD. The increased PPAR-γ protein in CYP4F2 transgenic mice fed HFD and the activation of rosiglitazone on the luciferase reporter construct of the NKCC2 promoter demonstrated that the increase in NKCC2 mRNA in CYP4F2 transgenic mice fed HFD was a consequence of elevated PPAR-γ protein induced by the synergy between 20-HETE and HFD., Conclusions: Our data demonstrated that the synergy between 20-HETE and HFD could decrease NKCC2 protein via posttranslational ubiquitination, which was thought to be the main mechanism underlying the short-term effect in response to HFD and might be responsible for the adaptive modulation of renal NKCC2 to resist sodium retention. Moreover, the increased NKCC2 mRNA expression via PPAR-γ-induced transcriptional regulation was thought to be the main mechanism underlying the long-term effect in response to HFD and plays a pivotal role in the development of obesity-related hypertension., (© 2021 The Author(s). Published by S. Karger AG, Basel.)

We conducted a multi-site investigation of genetic determinants of warfarin dose variability in Latinos from the U.S. and Brazil. Patients from four institutions in the United States (n = 411) and Brazil (n = 663) were genotyped for VKORC1 c.-1639G> A, common CYP2C9 variants, CYP4F2*3, and NQO1*2. Multiple regression analysis was used in the U.S. cohort to test the association between warfarin dose and genotype, adjusting for clinical factors, with further testing in an independent cohort of Brazilians. In the U.S. cohort, VKORC1 and CYP2C9 variants were associated with lower warfarin dose (β = -0.29, P < 2.0 × 10 -16 ; β = -0.21, P = 4.7 × 10 -7 , respectively) whereas CYP4F2 and NQO1 variants were associated with higher dose (β = 0.10, P = 2 × 10 -4 ; β = 0.10, P = 0.01, respectively). Associations with VKORC1 (β = -0.14, P = 2.0 × 10 -16 ), CYP2C9 (β = -0.07, P = 5.6 × 10 -10 ), and CYP4F2 (β = 0.03, P = 3 × 10 -3 ), but not NQO1*2 (β = 0.01, P = 0.30), were replicated in the Brazilians, explaining 43-46% of warfarin dose variability among the cohorts from the U.S. and Brazil, respectively. We identified genetic associations with warfarin dose requirements in the largest cohort of ancestrally diverse, warfarin-treated Latinos from the United States and Brazil to date. We confirmed the association of variants in VKORC1, CYP2C9, and CYP4F2 with warfarin dose in Latinos from the United States and Brazil., (© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society of Clinical Pharmacology and Therapeutics.)

Kaempferol has been confirmed to be effective in improving metabolic diseases such as diabetes and obesity. However, its effect and mechanism in nonalcoholic steatohepatitis (NASH) are unclear. We aim to confirm whether kaempferol could improve NASH and find the corresponding differential genes and metabolites. Transcriptomics combined with metabolomics was used to investigate the alterations in genes and metabolites expression after kaempferol treatment in mice with high-fat-diet-induced NASH. The results showed that kaempferol reduced the level of alanine transaminase (ALT), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) in serum and triglyceride (TG), lipid droplets, and inflammatory cell infiltration in liver. Further, 277 differentially expressed genes (DEGs) were identified through liver transcriptomics and the five most obvious DEGs were found to be CYP2b9, Cyp4a12b, Mup17, Mup7, and Mup16, which revealed that HFD induced fatty acid degradation, ribosome, and glyoxylic acid and dicarboxylic acid metabolism. Nine serum metabolites (methylcysteine, l-tryptophan, adrenic acid, d-2-hydroxyglutaric acid, tartaric acid, p-cresol sulfate, l-alanine, l-tryosine, and glutaconic acid) and 3 liver differential metabolites (gallic acid, γ-lindenic acid, and l-phenylalanine) were also identified, while the pathways were mainly involved in phenylalanine, tyrosine, and tryptophan biosynthesis; and phenylalanine metabolism. Integrating transcriptomics and metabolomics analyses indicated that kaempferol possesses the ability to improve NASH associated with energy metabolism, lipid metabolism, oxidative stress, and inflammation-related pathways. This study provides a powerful means of multiomics data integration and reveals the potent therapy and biomarkers for kaempferol.

Variation in levels of the human metabolome reflect changes in homeostasis, providing a window into health and disease. The genetic impact on circulating metabolites in Hispanics, a population with high cardiometabolic disease burden, is largely unknown. We conducted genome-wide association analyses on 640 circulating metabolites in 3,926 Hispanic Community Health Study/Study of Latinos participants. The estimated heritability for 640 metabolites ranged between 0%-54% with a median at 2.5%. We discovered 46 variant-metabolite pairs (p value < 1.2 × 10 -10 , minor allele frequency ≥ 1%, proportion of variance explained [PEV] mean = 3.4%, PEV range = 1%-22%) with generalized effects in two population-based studies and confirmed 301 known locus-metabolite associations. Half of the identified variants with generalized effect were located in genes, including five nonsynonymous variants. We identified co-localization with the expression quantitative trait loci at 105 discovered and 151 known loci-metabolites sets. rs5855544, upstream of SLC51A, was associated with higher levels of three steroid sulfates and co-localized with expression levels of SLC51A in several tissues. Mendelian randomization (MR) analysis identified several metabolites associated with coronary heart disease (CHD) and type 2 diabetes. For example, two variants located in or near CYP4F2 (rs2108622 and rs79400241, respectively), involved in vitamin E metabolism, were associated with the levels of octadecanedioate and vitamin E metabolites (gamma-CEHC and gamma-CEHC glucuronide); MR analysis showed that genetically high levels of these metabolites were associated with lower odds of CHD. Our findings document the genetic architecture of circulating metabolites in an underrepresented Hispanic/Latino community, shedding light on disease etiology., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Insect cuticle hydrocarbons are involved primarily in waterproofing the cuticle, but also participate in chemical communication and regulate the penetration of insecticides and microorganisms. The last step in insect hydrocarbon biosynthesis is carried out by an insect-specific cytochrome P450 of the 4G subfamily (CYP4G). Two genes (CYP4G106 and CYP4G107) have been reported in the triatomines Rhodnius prolixus and Triatoma infestans. In this work, their molecular and functional characterization is carried out in R. prolixus, and their relevance to insect survival is assessed. Both genes are expressed almost exclusively in the integument and have an expression pattern dependent on the developmental stage and feeding status. CYP4G106 silencing diminished significantly the straight-chain hydrocarbon production while a significant reduction - mostly of methyl-branched chain hydrocarbons - was observed after CYP4G107 silencing. Molecular docking analyses using different aldehydes as hydrocarbon precursors predicted a better fit of straight-chain aldehydes with CYP4G106 and methyl-branched aldehydes with CYP4G107. Survival bioassays exposing the silenced insects to desiccation stress showed that CYP4G107 is determinant for the waterproofing properties of the R. prolixus cuticle. This is the first report on the in vivo specificity of two CYP4Gs to make mostly straight or methyl-branched hydrocarbons, and also on their differential contribution to insect desiccation., (© 2020 The Royal Entomological Society.)

A series of N -acyl benzothiazoles shows selective and potent cytotoxicity against cancer cell lines expressing cytochrome P450 4F11. A prodrug form is metabolized by cancer cells into an active inhibitor of stearoyl-CoA desaturase (SCD). Substantial variation on the acyl portion of the inhibitors allowed the identification of ( R )- 27 , which balanced potency, solubility, and lipophilicity to allow proof-of-concept studies in mice. The prodrugs were activated inside the tumor, where they can arrest tumor growth. Together, these observations offer promise that a tumor-activated prodrug strategy might exploit the essentiality of SCD for tumor growth, while avoiding toxicity associated with systemic SCD inhibition.

Fingolimod is indicated for the treatment of patients with the relapsing-remitting form of multiple sclerosis. The primary study objective was to evaluate the bioequivalence of a test formulation, 0.5 mg fingolimod HCl capsule (Lebrina, Asofarma Sociedad Anónima Industrial y Comercial, Argentina) relative to a reference formulation, 0.5 mg fingolimod capsule (Gilenya, Novartis Pharmaceutical, Australia). In a single-center, randomized, single-dose, single-blinded, 2-way crossover study, 33 New Zealand healthy subjects of both sexes were enrolled to receive a 0.5-mg dose of 3 capsules of each fingolimod formulation under fasting conditions, with a 42-day washout period between administrations. Additional pharmacokinetic information regarding its main active metabolite, fingolimod phosphate, was also provided. The point estimate and 90% confidence intervals of the ratios of maximum concentration and area under the plasma concentration-time curve from time 0 to 72 hours were 99.07 (95.83-102.41) and 97.64 (95.33-100.00) for fingolimod, and 95.60 (90.95-100.49) and 98.54 (96.19-100.96), for fingolimod phosphate. Primary parameters, maximum concentration and area under the plasma concentration-time curve from time 0 to 72 hours for fingolimod and fingolimod phosphate were found to have no significant difference when test and reference formulations were compared. Fingolimod and fingolimod phosphate of both formulations were within the accepted 90% confidence interval limits of 80.00% and 125.00%. No significant differences between the test and reference drug products were detected in any of the pharmacokinetic parameters estimated. Notwithstanding the primary conclusion of bioequivalence is focused on the measurement of the parent compound, compliance with the same criteria by the active metabolite reinforces the comparability between the pharmacokinetic profiles of both formulations (ClinicalTrials.gov Identifier: NCT03757338)., (© 2020, The American College of Clinical Pharmacology.)

Cytochrome P450 monooxygenases (P450s) constitute a large superfamily of heme-thiolate proteins that are involved in the biosynthesis or degradation of endogenous compounds and detoxification of exogenous chemicals. It has been reported that P450s could serve as odorant-degrading enzymes (ODEs) to inactivate odorants to avoid saturating the antennae. However, there is little information about P450s in the antennae of Locusta migratoria. In the current work, we conducted an antenna transcriptome analysis and identified 92 P450s, including 68 full-length and 24 partial sequences. Phylogenetic analysis showed that 68 full-length P450s were grouped into four clans: CYP2, CYP3, CYP4, and mitochondria clans. Tissue, stage, and sex-dependent expressions of these 68 P450s were investigated. The results showed that 4 P450s were antenna-specific, whereas others were antenna-rich but also expressed in other tissues, implying their various potential roles in the antennae. In addition, the responses of seven selected P450s to five gramineous plant volatiles and four locust volatiles were determined. CYP6MU1 could be induced by almost all compounds tested, suggesting its important roles in odorant processing. Different P450s exhibited diverse responses to odorants, indicating that specific regulation of P450 expression by odorants might modulate the sensitivity of the olfactory responses to various chemicals., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The human cytochrome P450 enzyme CYP4Z1 remains an understudied enzyme despite its association with poor prognosis and overexpression in breast cancer. Hence, CYP4Z1 has previously been suggested as an anti-breast cancer target. In the present study we employed extended mutation analysis to increase our understanding of the substrate binding mode of this enzyme. In a combined in vitro and in silico approach we show for the first time that residue Arg487 plays an important role in substrate recognition and binding of CYP4Z1. Using a large array of recombinant CYP4Z1 mutants we show that, apart from Asn381, all other postulated binding residues only play an auxiliary role in substrate recognition and binding. Different substrate interaction motifs were identified via dynamic pharmacophores (dynophores) and their impact on catalytically competent substrate binding was classified. These new insights on the substrate recognition and binding mode represent an important step towards the rational design of CYP4Z1 prodrugs and guide further investigations into the so far poorly understood physiological role of CYP4Z1., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The skin permeability barrier is indispensable for maintaining water inside the body and preventing the invasion of pathogens and allergens; abnormalities lead to skin disorders such as atopic dermatitis and ichthyosis. Acylceramide is an essential lipid for skin barrier formation, and CYP4F22 is a fatty acid ω-hydroxylase involved in its synthesis. Mutations in CYP4F22 cause autosomal recessive congenital ichthyosis, although the symptoms vary among mutation sites and types. Here, we generated knockout mice deficient in Cyp4f39, the mouse ortholog of human CYP4F22, to investigate the effects of completely abrogating the function of the fatty acid ω-hydroxylase involved in acylceramide production on skin barrier formation. Cyp4f39 knockout mice died within 8 hours of birth. Large increases in transepidermal water loss and penetration of a dye from outside the body were observed, indicating severe skin barrier dysfunction. Histologic analyses of the epidermis revealed impairment of lipid lamella formation, accumulation of corneodesmosomes in the stratum corneum, and persistence of periderm. In addition, lipid analyses by mass spectrometry showed almost complete loss of acylceramide and its precursor ω-hydroxy ceramide. In conclusion, our findings provide clues to the molecular mechanisms of skin barrier abnormalities and the pathogenesis of ichthyosis caused by Cyp4f39 and CYP4F22 by association., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Fasting has been shown to regulate the expression of the cytochrome p450 (CYP) enzyme system in the liver. However, the exact mechanism behind the fasting-induced regulation of the CYP's remains unknown. In the present study we tested the hypothesis that the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), which is a key-regulator of energy metabolism, is responsible for the fasting-induced regulation of the CYP's. Lox/lox and liver specific PGC-1α (LKO) mice of both sexes, fasted for 18 h and the content of the CYP's as well as the hepatic metabolome was assessed. Fasting increased the mRNA content of Cyp2a4, Cyp2e1, Cyp3a11 and Cyp4a10. The fasting-induced response in Cyp4a10 mRNA content was different between lox/lox and LKO mice, while the absence of PGC-1α had no effect on the fasting-induced response for the other Cyp's. Moreover, the fasting-induced response in mRNA content of Sirtinus 1 and Perilipin 2 was different between lox/lox and LKO mice. Only the CYP1A isoform showed a fasting-induced response at the protein level. Absence of hepatic PGC-1α had no effect on the apparent metabolome, where fasting vs fed was the only discriminate in the following multivariate analysis. In conclusion, hepatic PGC-1α is not essential for the fasting-induced regulation of hepatic CYP's., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sesamin is a major lignan in sesame seeds, and a recent meta-analysis of controlled trials indicated that sesamin intake decreases blood pressure. The antihypertensive effect of sesamin has been suggested to be due to sesamin-mediated suppression of 20-hydroxyeicosatetraenoic acid production catalyzed by CYP4F2. However, the detailed mechanism underlying inhibition of CYP4F2 function by sesamin remains unclear. In this study, the effects of sesamin on catalytic activity of CYP4F2 were investigated in vitro. Sesamin inhibited luciferin-4F2/3 O-dealkylase activity of recombinant human CYP4F2 with an IC 50 value of 0.381 µM. When preincubated in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) for 20 min, sesamin potentiated the inhibition of CYP4F2 activity. Moreover, kinetic analysis of the inactivation revealed that sesamin showed a preincubation time- and concentration-dependent inhibition of CYP4F2 activity yielding a maximal inactivation rate constant (k inact ) value of 0.354 min -1 and half-maximal inhibitory concentration (K I ) value of 1.12 µM. The inactivation of CYP4F2 by sesamin required NADPH. These results indicated that sesamin is a mechanism-based inactivator of human CYP4F2.

Our previous studies have shown that α-eleostearic acid (α-ESA; cis-9, trans-11, trans-13 (c9,t11,t13)-conjugated linolenic acid (CLnA)) is converted into c9,t11-conjugated linoleic acid (CLA) in rats. Furthermore, we have demonstrated that the conversion of α-ESA into CLA is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzymatic reaction, which occurs mostly in the rat liver. However, the precise metabolic pathway and enzyme involved have not been identified yet. Therefore, in this study we aimed to determine the role of cytochrome P450 (CYP) in the conversion of α-ESA into c9,t11-CLA using an in vitro reconstitution system containing mouse hepatic microsomes, NADPH, and α-ESA. The CYP4 inhibitors, 17-ODYA and HET0016, performed the highest level of inhibition of CLA formation. Furthermore, the redox partner cytochrome P450 reductase (CPR) inhibitor, 2-chloroethyl ethyl sulfide (CEES), also demonstrated a high level of inhibition. Thus, these results indicate that the NADPH-dependent CPR/CYP4 system is responsible for CLA formation. In a correlation analysis between the specific activity of CLA formation and Cyp4 family gene expression in tissues, Cyp4a14 and Cyp4f13 demonstrated the best correlations. However, the CYP4F substrate prostaglandin A 1 (PGA 1 ) exhibited the strongest inhibitory effect on CLA formation, while the CYP4A and CYP4B1 substrate lauric acid had no inhibitory effect. Therefore, we conclude that the CYP4F13 enzyme is the major enzyme involved in CLA formation. This pathway is a novel pathway for endogenous CLA synthesis, and this study provides insight into the potential application of CLnA in functional foods.