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BACKGROUND: Recent biological studies have demonstrated that changes can occur in the cellular genome and proteome due to variations in cell volume. Therefore, it is imperative to take cell volume into account when analyzing a target protein. This consideration becomes especially critical in experimental models involving cells subjected to different treatments. Failure to consider cell volume could obscure the studied biological phenomena or lead to erroneous conclusions. However, quantitative imaging of proteins within cells by LA-ICP-MS is limited by the lack of methods that provide the protein concentration (protein mass over cell volume) rather than just protein mass within individual cells. RESULTS: The combination of a metal tagged immunoprobe with ruthenium red (RR) labelling enables the simultaneous analysis of a specific protein and the cell volume in each cell analyzed by LA-ICP-(Q)MS. The results indicate that the CYP1B1 concentration exhibits a quasi-normally distribution in control ARPE-19 cells, whereas AAPH-treated cells reveal the presence of two distinct cell groups, responding and non-responding cells to an in vitro induced oxidative stress. The labelling of the membrane with RR and the measurement of Ru mass in each cell by LA-ICP-MS offers higher precision compared to manually delimitation of the cell perimeter and eliminates the risk of biased information, which can be prone to inter-observer variability. The proposed procedure is fast and minimizes errors in cell area assignment and offers the possibility to carry out a faster data treatment approach if just relative volumes are compared, which can be advantageous for specific applications. SIGNIFICANCE AND NOVELTY: This work presents an innovative strategy to directly study the distribution and concentration of proteins within individual cells by LA-ICP-MS. This method employs ruthenium red as a cell volume marker and Au nanoclusters (AuNCs) tagged immunoprobes to label the protein of interest. Furthermore, the proposed labelling strategy enables rapid data processing, allowing for the calculation of relative concentrations and thus facilitating the comparison across large datasets. As a proof-of-concept, the concentration of the CYP1B1 protein was quantified in ARPE-19 cells under both control and oxidative stress conditions.
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Espectrometría de Masas , Nanopartículas del Metal , Humanos , Nanopartículas del Metal/química , Citocromo P-450 CYP1B1/metabolismo , Línea Celular , Estrés OxidativoRESUMEN
BACKGROUND: Primary congenital glaucoma (PCG) affects approximately 1 in 10,000 live born infants in the United States (U.S.). PCG has a autosomal recessive inheritance pattern, and variable expressivity and reduced penetrance have been reported. Likely causal variants in the most commonly mutated gene, CYP1B1, are less prevalent in the U.S., suggesting that alternative genes may contribute to the condition. This study utilized exome sequencing to investigate the genetic architecture of PCG in the U.S. and to identify novel genes and variants. METHODS: We studied 37 family trios where infants had PCG and were part of the National Birth Defects Prevention Study (births 1997-2011), a U.S. multicenter study of birth defects. Samples underwent exome sequencing and sequence reads were aligned to the human reference sample (NCBI build 37/hg19). Variant filtration was conducted under de novo and Mendelian inheritance models using GEMINI. RESULTS: Among candidate variants, CYP1B1 was most represented (five trios, 13.5%). Twelve probands (32%) had potentially pathogenic variants in other genes not previously linked to PCG but important in eye development and/or to underlie Mendelian conditions with potential phenotypic overlap (e.g., CRYBB2, RXRA, GLI2). CONCLUSION: Variation in the genes identified in this population-based study may help to further explain the genetics of PCG.
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Citocromo P-450 CYP1B1 , Secuenciación del Exoma , Exoma , Glaucoma , Humanos , Glaucoma/genética , Glaucoma/congénito , Citocromo P-450 CYP1B1/genética , Femenino , Masculino , Secuenciación del Exoma/métodos , Estados Unidos , Exoma/genética , Mutación/genética , Predisposición Genética a la Enfermedad , Lactante , Recién NacidoRESUMEN
This research aimed to clarify the impacts of cannflavin-C on angiotensin II (Ang II)-induced cardiac hypertrophy and their potential role in modulating cytochrome P450 1B1 (CYP1B1) and arachidonic acid (AA) metabolites. Currently there is no evidence to suggest that cannflavin-C, a prenylated flavonoid, has any significant effects on the heart or cardiac hypertrophy. The metabolism of arachidonic acid (AA) into midchain hydroxyeicosatetraenoic acids (HETEs), facilitated by CYP1B1 enzyme, plays a role in the development of cardiac hypertrophy, which is marked by enlarged cardiac cells. Adult human ventricular cardiomyocyte (AC16) cell line was cultured and exposed to cannflavin-C in the presence and absence of Ang II. The assessment of mRNA expression pertaining to cardiac hypertrophic markers and cytochromes P450 (P450s) was conducted via real-time polymerase chain reaction (PCR), whereas the quantification of P450 protein levels was carried out through western blot analysis. Ang II induced hypertrophic markers myosin heavy chain (ß/α-MHC), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) and increased cell surface area, whereas cannflavin-C mitigated these effects. Gene and protein expression analysis revealed that cannflavin-C downregulated CYP1B1 gene expression, protein level, and enzyme activity assessed by 7-methoxyresorufin O-deethylase (MROD). Arachidonic acid metabolites analysis, using liquid chromatography-tandem mass spectrometry (LC-MS/MS), demonstrated that Ang II increased midchain (R/S)-HETE concentrations, which were attenuated by cannflavin-C. This study provides novel insights into the potential of cannflavin-C in modulating arachidonic acid metabolites and attenuating Ang II-induced cardiac hypertrophy, highlighting the importance of this compound as potential therapeutic agents for cardiac hypertrophy. SIGNIFICANCE STATEMENT: This study demonstrates that cannflavin-C offers protection against cellular hypertrophy induced by angiotensin II. The significance of this research lies in its novel discovery, which elucidates a mechanistic pathway involving the inhibition of CYP1B1 by cannflavin-C. This discovery opens up new avenues for leveraging this compound in the treatment of heart failure.
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Angiotensina II , Ácido Araquidónico , Cardiomegalia , Citocromo P-450 CYP1B1 , Miocitos Cardíacos , Citocromo P-450 CYP1B1/metabolismo , Citocromo P-450 CYP1B1/genética , Angiotensina II/farmacología , Angiotensina II/toxicidad , Humanos , Ácido Araquidónico/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/inducido químicamente , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Línea Celular , Ácidos Hidroxieicosatetraenoicos/metabolismoRESUMEN
Within the sequence of the CYP1B1 gene, more than 50 polymorphisms, resulting from single-nucleotide polymorphisms (SNPs), have been described. Some of them play an important role as specific genetic markers in the process of carcinogenesis and for therapeutic purposes. In this publication, we present methods we have developed that enable the specific and unambiguous identification of four polymorphisms that result in amino acid changes: c. 142C > G, c. 355G > T, c. 1294C > G, and c. 1358A > G. Our studies are based on cleaved amplified polymorphic sequences (CAPSs) and artificially created restriction site (ACRS) PCR techniques; therefore, they require only basic laboratory equipment and low financial outlays. Utilizing the described methods allows for the reduction of research time and cost, and the minimization of errors. Their effectiveness and efficiency depend on the careful design of appropriate primers and the precise selection of suitable restriction enzymes. As a result, further confirmation by sequencing is not necessary. Using the developed method, we examined 63 patients diagnosed with lung cancer and observed a 1.5 to 2.1 times higher frequency of the analyzed single-nucleotide polymorphisms compared to the frequency in the European population.
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Citocromo P-450 CYP1B1 , Neoplasias Pulmonares , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Humanos , Citocromo P-450 CYP1B1/genética , Neoplasias Pulmonares/genética , Reacción en Cadena de la Polimerasa/métodos , Femenino , Masculino , Persona de Mediana Edad , AncianoRESUMEN
Heart failure (HF) is preceded by cellular hypertrophy (CeH) which alters expression of cytochrome P450 enzymes (CYPs) and arachidonic acid (AA) metabolism. Inflammation is involved in CeH pathophysiology, but mechanisms remain elusive. This study investigates the impacts of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and lipopolysaccharides (LPS) on the development of CeH and the role of CYP1B1. AC16 cells were treated with TNF-α, IL-6, and LPS in the presence and absence of CYP1B1-siRNA or resveratrol. mRNA and protein expression levels of CYP1B1 and hypertrophic markers were determined using PCR and Western blot analysis, respectively. CYP1B1 enzyme activity was determined, and AA metabolites were analyzed using liquid chromatography-tandem mass spectrometry. Our results show that TNF-α, IL-6, and LPS induce expression of hypertrophic markers, induce CYP1B1 expression, and enantioselectively modulate CYP1B1-mediated AA metabolism in favor of mid-chain HETEs. CYP1B1-siRNA or resveratrol ameliorated these effects. In conclusion, our results demonstrate the crucial role of CYP1B1 in TNF-α, IL-6, and LPS-induced CeH.
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Citocromo P-450 CYP1B1 , Interleucina-6 , Lipopolisacáridos , Resveratrol , Factor de Necrosis Tumoral alfa , Humanos , Línea Celular , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Interleucina-6/metabolismo , Interleucina-6/genética , Resveratrol/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Human cytochrome P450 1B1 enzyme (hCYP1B1), a member of hCYP1 subfamily, plays a crucial role in multiple diseases by participating in many metabolic pathways. Although a suite of potent hCYP1B1 inhibitors have been previously reported, most of them also act as aryl hydrocarbon receptor (AhR) agonists that can up-regulate the expression of hCYP1B1 and then counteract their inhibitory potential in living systems. This study aimed to develop novel efficacious hCYP1B1 inhibitors that worked well in living cells but without AhR agonist effects. For these purposes, a series of 1,8-naphthalimide derivatives were designed and synthesized, and their structure-activity relationships (SAR) as hCYP1B1 inhibitors were analyzed. Following three rounds SAR studies, several potent hCYP1B1 inhibitors were discovered, among which compound 3n was selected for further investigations owing to its extremely potent anti-hCYP1B1 activity (IC50 = 0.040 nM) and its blocking AhR transcription activity in living cells. Inhibition kinetic analyses showed that 3n potently inhibited hCYP1B1 via a mix inhibition manner, showing a Ki value of 21.71 pM. Docking simulations suggested that introducing a pyrimidine moiety to the hit compound (1d) facilitated 3n to form two strong interactions with hCYP1B1/heme, viz., the C-Brâ¯π halogen bond and the N-Fe coordination bond. Further investigations demonstrated that 3n (5 µM) could significantly reverse the paclitaxel (PTX) resistance in H460/PTX cells, evidenced by the dramatically reduced IC50 values, from 632.6 nM (PTX alone) to 100.8 nM (PTX plus 3n). Collectively, this study devised a highly potent hCYP1B1 inhibitor (3n) without AhR agonist effect, which offered a promising drug candidate for overcoming hCYP1B1-associated drug resistance.
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Citocromo P-450 CYP1B1 , Diseño de Fármacos , Naftalimidas , Humanos , Relación Estructura-Actividad , Naftalimidas/farmacología , Naftalimidas/química , Naftalimidas/síntesis química , Citocromo P-450 CYP1B1/antagonistas & inhibidores , Citocromo P-450 CYP1B1/metabolismo , Estructura Molecular , Relación Dosis-Respuesta a DrogaRESUMEN
Childhood glaucoma (CG) encompasses a heterogeneous group of genetic eye disorders that is responsible for approximately 5% of childhood blindness worldwide. Understanding the molecular aetiology is key to improving diagnosis, prognosis and unlocking the potential for optimising clinical management. In this study, we investigated 86 CG cases from 78 unrelated families of diverse ethnic backgrounds, recruited into the Genomics England 100,000 Genomes Project (GE100KGP) rare disease cohort, to improve the genetic diagnostic yield. Using the Genomics England/Genomic Medicine Centres (GE/GMC) diagnostic pipeline, 13 unrelated families were solved (13/78, 17%). Further interrogation using an expanded gene panel yielded a molecular diagnosis in 7 more unrelated families (7/78, 9%). This analysis effectively raises the total number of solved CG families in the GE100KGP to 26% (20/78 families). Twenty-five percent (5/20) of the solved families had primary congenital glaucoma (PCG), while 75% (15/20) had secondary CG; 53% of this group had non-acquired ocular anomalies (including iris hypoplasia, megalocornea, ectopia pupillae, retinal dystrophy, and refractive errors) and 47% had non-acquired systemic diseases such as cardiac abnormalities, hearing impairment, and developmental delay. CYP1B1 was the most frequently implicated gene, accounting for 55% (11/20) of the solved families. We identified two novel likely pathogenic variants in the TEK gene, in addition to one novel pathogenic copy number variant (CNV) in FOXC1. Variants that passed undetected in the GE100KGP diagnostic pipeline were likely due to limitations of the tiering process, the use of smaller gene panels during analysis, and the prioritisation of coding SNVs and indels over larger structural variants, CNVs, and non-coding variants.
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Glaucoma , Humanos , Glaucoma/genética , Glaucoma/diagnóstico , Masculino , Femenino , Niño , Preescolar , Citocromo P-450 CYP1B1/genética , Mutación , Lactante , Genómica/métodos , Linaje , Adolescente , Factores de Transcripción ForkheadRESUMEN
Importance: Frontal fibrosing alopecia (FFA) is an increasingly prevalent form of follicular lichen planus, causing irreversible hair loss predominantly in postmenopausal individuals. An earlier genome-wide meta-analysis of female FFA identified risk loci in genes implicated in self-antigen presentation and T-cell homeostasis, including HLA-B*07:02, ST3GAL1, and SEMA4B. However, CYP1B1, which is important for hormone metabolism, was also implicated with the substitution of serine for asparagine at position 453 (c.1358A>G, p.Asn453Ser) exhibiting a protective effect against FFA. Increasing understanding of genetic and environmental variables and their interactions will improve understanding of disease pathogenesis and has the potential to inform risk mitigation strategies. Objective: To investigate whether oral contraceptive pill (OCP) use modulates the protective effect of the common missense variant in CYP1B1 (c.1358A>G, p.Asn453Ser) on FFA risk. Design, Setting, and Participants: This gene-environment interaction study using a case-control design enrolled female patients with FFA from UK-based dermatology clinics. The patients were matched with unrelated age- and ancestry-matched female control individuals derived from UK Biobank in a 1:66 ratio, determined by the first 4 principal components from genome-wide genotypes. Data were collected from July 2015 to September 2017, and analyzed from October 2022 to December 2023. Main Outcome and Measure: The main outcomes were the modulatory effect of OCP use on the contribution of the CYP1B1 missense variant to female FFA risk and a formal gene-environment interaction test evaluated by a logistic regression model with a multiplicative interaction term, under the assumptions of an additive genetic model interaction term, under the assumptions of an additive genetic model. Results: Of the 489 female patients with FFA, the mean (SD) age was 65.8 (9.7) years, and 370 (75.7%) had a history of OCP use. Of the 34â¯254 age- and ancestry-matched control individuals, the mean (SD) age was 65.0 (8.4) years, and previous OCP use was reported in 31â¯177 (91.0%). An association between female FFA and the CYP1B1 risk allele was observed in individuals who reported OCP use (odds ratio, 1.90 [95% CI, 1.50-2.40]; P = 8.41 × 10-8) but not in those with no documented exposure to OCPs (odds ratio, 1.16 [95% CI, 0.82-1.64]; P = .39). A full gene-environment interaction model demonstrated a significant additive statistical interaction between c.1358A, p.453Asn, and history of OCP use on FFA risk (OR for interaction, 1.63 [95% CI, 1.07-2.46]; P = .02). Conclusions and Relevance: This gene-environment interaction analysis suggests that the protective effect of the CYP1B1 missense variant on FFA risk might be mediated by exposure to OCPs. The allele that encodes an asparagine at position 453 of CYP1B1 was associated with increased odds of FFA only in participants with OCP history.
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Alopecia , Citocromo P-450 CYP1B1 , Interacción Gen-Ambiente , Humanos , Femenino , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Alopecia/genética , Persona de Mediana Edad , Estudios de Casos y Controles , Anticonceptivos Orales/efectos adversos , Anticonceptivos Orales/administración & dosificación , Anciano , Adulto , Predisposición Genética a la Enfermedad , Liquen Plano/genética , Mutación Missense , Reino Unido/epidemiologíaRESUMEN
The prevalence of fragrances in various hygiene products contributes to their sensorial allure. However, fragrances can induce sensitization in the skin or respiratory system, and the mechanisms involved in this process are incompletely understood. This study investigated the intricate mechanisms underlying the fragrance's effects on sensitization response, focusing on the interplay between CYP450 enzymes, a class of drug-metabolizing enzymes, and the adaptive immune system. Specifically, we assessed the expression of CYP450 enzymes and cytokine profiles in culture of BEAS-2B and mature dendritic cells (mDC) alone or in co-culture stimulated with 2 mM of a common fragrance, cinnamyl alcohol (CA) for 20 h. CYP1A1, CYP1A2, CYP1B1, CYP2A6, and CYP2A13 were analyzed by RT-PCR and IL-10, IL-12p70, IL-18, IL-33, and thymic stromal lymphopoietin (TSLP) by Cytometric Bead Array (CBA). Through RT-PCR analysis, we observed that CA increased CYP1A2 and CYP1B1 expression in BEAS-2B, with a further increased in BEAS-2B-mDC co-culture. Additionally, exposure to CA increased IL-12p70 levels in mDC rather than in BEAS-2B-mDC co-culture. In regards to IL-18, level was higher in BEAS-2B than in BEAS-2B-mDC co-culture. A positive correlation between the levels of IL-10 and CYP1B1 was found in mDC-CA-exposed and between IL-12p70 and CYP1A1 was found in BEAS-2B after CA exposure. However, IL-12p70 and CYP1A2 as well as IL-18, IL-33, and CYP1A1 levels were negative, correlated mainly in co-culture control. These correlations highlight potential immunomodulatory interactions and complex regulatory relationships. Overall, exposure to CA enhances CYP450 expression, suggesting that CA can influence immune responses by degrading ligands on xenosensitive transcription factors.
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Técnicas de Cocultivo , Sistema Enzimático del Citocromo P-450 , Citocinas , Células Dendríticas , Propanoles , Humanos , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Propanoles/toxicidad , Propanoles/metabolismo , Línea Celular , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Perfumes/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Hidrocarburo de Aril/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1A2/genéticaRESUMEN
Extrahepatic cytochrome P450 1B1 (CYP1B1), which is highly expressed in non-small cell lung cancer, is an attractive target for cancer prevention, therapy, and overcoming drug resistance. Historically, CYP1B1 inhibition has been the primary therapeutic approach for treating CYP1B1-related malignancies, but its success has been limited. This study introduced CYP1B1 degradation as an alternative strategy to counter drug resistance and metastasis in CYP1B1-overexpressing non-small cell lung cancer A549/Taxol cells via a PROTAC strategy. Our investigation revealed that the identification of the potent CYP1B1 degrader PV2, achieving DC50 values of 1.0 nM and inducing >90 % CYP1B1 degradation at concentrations as low as 10 nM in A549/Taxol cells. Importantly, PV2 enhanced the sensitivity of the A549/Taxol subline to Taxol, possibly due to its stronger inhibitory effects on P-gp through CYP1B1 degradation. Additionally, compared to the CYP1B1 inhibitor A1, PV2 effectively suppressed the migration and invasion of A549/Taxol cells by inhibiting the FAK/SRC and EMT pathways. These findings hold promise for a novel therapy targeting advanced CYP1B1+ non-small cell lung cancer.
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Antineoplásicos , Citocromo P-450 CYP1B1 , Resistencia a Antineoplásicos , Citocromo P-450 CYP1B1/antagonistas & inhibidores , Citocromo P-450 CYP1B1/metabolismo , Humanos , Resistencia a Antineoplásicos/efectos de los fármacos , Estructura Molecular , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Relación Estructura-Actividad , Ensayos de Selección de Medicamentos Antitumorales , Relación Dosis-Respuesta a Droga , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Movimiento Celular/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Paclitaxel/farmacología , Paclitaxel/química , Tiazoles/química , Tiazoles/farmacología , Tiazoles/síntesis químicaRESUMEN
Chronic kidney disease (CKD) causes accumulation of uremic metabolites that negatively affect skeletal muscle. Tryptophan-derived uremic metabolites are agonists of the aryl hydrocarbon receptor (AHR), which has been shown to be activated in CKD. This study investigated the role of the AHR in skeletal muscle pathology of CKD. Compared with controls with normal kidney function, AHR-dependent gene expression (CYP1A1 and CYP1B1) was significantly upregulated in skeletal muscle of patients with CKD, and the magnitude of AHR activation was inversely correlated with mitochondrial respiration. In mice with CKD, muscle mitochondrial oxidative phosphorylation (OXPHOS) was markedly impaired and strongly correlated with the serum level of tryptophan-derived uremic metabolites and AHR activation. Muscle-specific deletion of the AHR substantially improved mitochondrial OXPHOS in male mice with the greatest uremic toxicity (CKD + probenecid) and abolished the relationship between uremic metabolites and OXPHOS. The uremic metabolite/AHR/mitochondrial axis in skeletal muscle was verified using muscle-specific AHR knockdown in C57BL/6J mice harboring a high-affinity AHR allele, as well as ectopic viral expression of constitutively active mutant AHR in mice with normal renal function. Notably, OXPHOS changes in AHRmKO mice were present only when mitochondria were fueled by carbohydrates. Further analyses revealed that AHR activation in mice led to significantly increased pyruvate dehydrogenase kinase 4 (Pdk4) expression and phosphorylation of pyruvate dehydrogenase enzyme. These findings establish a uremic metabolite/AHR/Pdk4 axis in skeletal muscle that governs mitochondrial deficits in carbohydrate oxidation during CKD.
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Ratones Endogámicos C57BL , Músculo Esquelético , Fosforilación Oxidativa , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Receptores de Hidrocarburo de Aril , Insuficiencia Renal Crónica , Triptófano , Animales , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Hidrocarburo de Aril/genética , Ratones , Masculino , Insuficiencia Renal Crónica/metabolismo , Triptófano/metabolismo , Músculo Esquelético/metabolismo , Humanos , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/genética , Uremia/metabolismo , Mitocondrias Musculares/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Femenino , Ratones Noqueados , Citocromo P-450 CYP1B1/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A1/genética , Persona de Mediana Edad , Metabolismo Energético , Modelos Animales de EnfermedadRESUMEN
Targeting circular RNA has been a novel approach to preventing and limiting acute myocardial infarction (AMI). Here, we planned to investigate the role and mechanism of circ_0020887 in AMI progression.Hypoxic injury in human cardiomyocytes (AC16) was measured using cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine assay, flow cytometry, and colorimetric assay kits. RNA and protein expressions were determined using real-time quantitative PCR and western blotting. Direct interplay between RNAs was determined using dual-luciferase reporter, RNA pull-down, and RIP assays.In the plasma and hypoxia-induced AC16 cells of patients with AMI, circ_0020887 and miR-370-3p were upregulated and downregulated, respectively, concomitant with the upregulation of cytochrome P450 1B1 (CYP1B1). Circ_0020887 interference could inhibit hypoxia-induced AC16 cell apoptosis, oxidative stress, and inflammatory response. Circ_0020887 could sponge miR-370-3p, and miR-370-3p could target CYP1B1. The inhibition effect of circ_0020887 knockdown on hypoxia-induced AC16 cell injury could be reversed by the miR-370-3p inhibitor. Besides, CYP1B1 overexpression also overturned the suppressive effect of miR-370-3p on hypoxia-induced AC16 cell apoptosis, oxidative stress, and inflammatory response.In conclusion, circ_0020887 regulated the miR-370-3p/CYP1B1 axis to regulate hypoxia-induced cardiomyocyte injury, confirming that circ_0020887 might promote cardiomyocyte injury.
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MicroARNs , Infarto del Miocardio , Humanos , Miocitos Cardíacos , Apoptosis/genética , Western Blotting , Hipoxia , MicroARNs/genética , Proliferación Celular , Citocromo P-450 CYP1B1RESUMEN
BACKGROUND: Cytochrome P450 (CYP) comprises a group of phase-I metabolizing enzymes that are important in xenobiotics metabolism. Genetic polymorphism of CYPs has been comprehensively studied for their association with a range of diseases. In this study, we assessed single-nucleotide polymorphism (SNP) of CYP1A, CYP1B, CYP2B, and CYP2C and their role in gastrointestinal (GI) cancer susceptibility in the rural population of Maharashtra. MATERIALS AND METHODS: In this hospital-based case-control study, the association of polymorphism of CYP genes was studied by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The study subjects included 200 clinically confirmed GI cancer patients and equal number of healthy controls. Odds ratio (OR) with 95% confidence interval (CI) and P value were evaluated to find out the level of association, where P ≤ 0.005 was considered statistically significant. RESULTS: After the analysis of CYP1A1*2A (rs4646903), CYP1B1*3 (rs1059836), CYP2B6*5 (rs3211371), CYP2C8*2 (rs11572103), CYP2C9*2 (rs1799853), and CYP2C9*3 (rs1057910), we noticed that variant (T) allele of CYP2B6*5 possessed significantly elevated risk (OR = 4.43; 95% CI: 2.20-8.90; P < 0.0001) of GI cancer in studied population. The genotypic distribution of G/C heterozygote allele of CYP1B1*3 (OR = 0.19, 95% CI = 0.12-0.32; P < 0.0001) and homozygous variant C/C allele (OR = 0.24, 95% CI = 0.13-0.45; P < 0.0001) showed a negative association with the development of GI cancer. CONCLUSION: The findings from this study supported that polymorphism of CYP2B6*5gene may be involved in the development of GI cancer. However, other SNPs of CYP1A, CYP1B, and CYP2C genes did not signify the risk for GI cancer in the studied population of rural Maharashtra.
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Citocromo P-450 CYP1A1 , Neoplasias Gastrointestinales , Humanos , Citocromo P-450 CYP1A1/genética , Polimorfismo de Nucleótido Simple , Citocromo P-450 CYP2C8/genética , Citocromo P-450 CYP2C9/genética , Estudios de Casos y Controles , Citocromo P-450 CYP2B6/genética , India/epidemiología , Genotipo , Neoplasias Gastrointestinales/genética , Citocromo P-450 CYP1B1/genéticaRESUMEN
Cytochrome P450 1B1 (CYP1B1) contributes to the metabolic inactivation of chemotherapeutics when overexpressed in tumor cells. Selective inhibition of CYP1B1 holds promise for reversing drug resistance. In our pursuit of potent CYP1B1 inhibitors, we designed and synthesized a series of 2-phenylquinazolin-4-amines. A substantial proportion of these newly developed inhibitors demonstrated inhibitory activity against CYP1B1, accompanied by improved water solubility. Remarkably, compound 14b exhibited exceptional inhibitory efficacy and selectivity toward CYP1B1. Molecular docking studies suggested that the expansion of the π-system through aromatization, the introduction of an amine group, and iodine atom augmented the binding affinity. Furthermore, inhibitors 14a, 14b, and 14e demonstrated the ability to significantly reduce the resistance in A549 cells to paclitaxel, while also inhibiting the migration and invasion of these cells. Finally, radioiodine labeling experiments shed light on the metabolic pathway of compound 5l in mice, highlighting the potential of 125I-5l as a radioactive probe for future research endeavors.
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Radioisótopos de Yodo , Paclitaxel , Animales , Ratones , Humanos , Paclitaxel/farmacología , Células A549 , Simulación del Acoplamiento Molecular , Aminas , Citocromo P-450 CYP1B1/químicaRESUMEN
AIMS: Excessive influx of manganese (Mn) into the brain across the blood-brain barrier induces neurodegeneration. CYP1B1 is involved in the metabolism of arachidonic acid (AA) that affects vascular homeostasis. We aimed to investigate the effect of brain CYP1B1 on Mn-induced neurotoxicity. METHOD: Brain Mn concentrations and α-synuclein accumulation were measured in wild-type and CYP1B1 knockout mice treated with MnCl2 (30 mg/kg) and biotin (0.2 g/kg) for 21 continuous days. Tight junctions and oxidative stress were analyzed in hCMEC/D3 and SH-SY5Y cells after the treatment with MnCl2 (200 µM) and CYP1B1-derived AA metabolites (HETEs and EETs). RESULTS: Mn exposure inhibited brain CYP1B1, and CYP1B1 deficiency increased brain Mn concentrations and accelerated α-synuclein deposition in the striatum. CYP1B1 deficiency disrupted the integrity of the blood-brain barrier (BBB) and increased the ratio of 3, 4-dihydroxyphenylacetic acid (DOPAC) to dopamine in the striatum. HETEs attenuated Mn-induced inhibition of tight junctions by activating PPARγ in endothelial cells. Additionally, EETs attenuated Mn-induced up-regulation of the KLF/MAO-B axis and down-regulation of NRF2 in neuronal cells. Biotin up-regulated brain CYP1B1 and reduced Mn-induced neurotoxicity in mice. CONCLUSIONS: Brain CYP1B1 plays a critical role in both cerebrovascular and dopamine homeostasis, which might serve as a novel therapeutic target for the prevention of Mn-induced neurotoxicity.
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Barrera Hematoencefálica , Citocromo P-450 CYP1B1 , Neuroblastoma , Animales , Humanos , Ratones , alfa-Sinucleína/metabolismo , Biotina/metabolismo , Barrera Hematoencefálica/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Dopamina/metabolismo , Células Endoteliales/metabolismo , Manganeso/toxicidad , Estrés OxidativoRESUMEN
Inhalation exposure to plastic incineration emissions (PIEs) is a problem of increasing human relevance, as plastic production and waste creation have drastically increased since mainstream integration during the 20th century. We investigated the effects of PIEs on human nasal epithelial cells (HNECs) to understand if such exposures cause damage and dysfunction to respiratory epithelia. Primary HNECs from male and female donors were cultured at air-liquid interface (ALI), and 16HBE cells were cultured on coverslips. Smoke condensates were generated from incineration of plastic at flaming (640°C) and smoldering (500°C) temperatures, and cells were subsequently exposed to these materials at 5-50 µg/cm2 concentrations. HNECs were assessed for mitochondrial dysfunction and 16HBE cells for glutathione oxidation in real-time analyses. HNEC culture supernatants and total RNA were collected at 4-h postexposure for cytokine and gene expression analysis, and results show that PIEs can acutely induce inflammation, oxidative stress, and mitochondrial dysfunction in HNECs, and that incineration temperature modifies biological responses. Specifically, condensates from flaming and smoldering PIEs significantly increased HNEC secretion of cytokines IL-8, IL-1ß, and IL-13, as well as expression of xenobiotic metabolism pathways and genes such as CYP1A1 and CYP1B1 at 5 and 20 µg/cm2 concentrations. Only 50 µg/cm2 flaming PIEs significantly increased glutathione oxidation in 16HBEs, and decreased respiration and ATP production in HNEC mitochondria. Impact Statement: Our data reveal the impact of incineration temperatures on biological outcomes associated with PIE exposures, emphasizing the importance of temperature as a factor when evaluating respiratory disease associated with PIEs exposure.
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Contaminantes Atmosféricos , Células Epiteliales , Incineración , Inflamación , Estrés Oxidativo , Humanos , Estrés Oxidativo/efectos de los fármacos , Femenino , Masculino , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Contaminantes Atmosféricos/toxicidad , Inflamación/inducido químicamente , Inflamación/metabolismo , Plásticos/toxicidad , Metabolismo Energético/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Glutatión/metabolismo , Humo/efectos adversos , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Exposición por Inhalación/efectos adversosRESUMEN
BACKGROUND: Renal cell carcinoma (RCC) is one of the most common malignant tumor worldwide. Metastasis is a leading case of cancer-related deaths of RCC. Circular RNAs (circRNAs), a class of noncoding RNAs, have emerged as important regulators in cancer metastasis. However, the functional effects and regulatory mechanisms of circRNAs on RCC metastasis remain largely unknown. METHODS: High-throughput RNA sequencing techniques were performed to analyze the expression profiles of circRNAs and mRNAs in highly and poorly invasive clear cell renal cell carcinoma (ccRCC) cell lines. Functional experiments were performed to unveil the regulatory role of circPPAP2B in the proliferation and metastatic capabilities of ccRCC cells. RNA pulldown, Mass spectrometry analysis, RNA methylation immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), co-immunoprecipitation (CoIP), next-generation RNA-sequencing and double luciferase experiments were employed to clarify the molecular mechanisms by which circPPAP2B promotes ccRCC metastasis. RESULTS: In this study, we describe a newly identified circular RNA called circPPAP2B, which is overexpressed in highly invasive ccRCC cells, as determined through advanced high-throughput RNA sequencing techniques. Furthermore, we observed elevated circPPAP2B in ccRCC tissues, particularly in metastatic ccRCC tissues, and found it to be associated with poor prognosis. Functional experiments unveiled that circPPAP2B actively stimulates the proliferation and metastatic capabilities of ccRCC cells. Mechanistically, circPPAP2B interacts with HNRNPC in a m6A-dependent manner to facilitate HNRNPC nuclear translocation. Subcellular relocalization was dependent upon nondegradable ubiquitination of HNRNPC and stabilization of an HNRNPC/Vimentin/Importin α7 ternary complex. Moreover, we found that circPPAP2B modulates the interaction between HNRNPC and splicing factors, PTBP1 and HNPNPK, and regulates pre-mRNA alternative splicing. Finally, our studies demonstrate that circPPAP2B functions as a miRNA sponge to directly bind to miR-182-5p and increase CYP1B1 expression in ccRCC. CONCLUSIONS: Collectively, our study provides comprehensive evidence that circPPAP2B promotes proliferation and metastasis of ccRCC via HNRNPC-dependent alternative splicing and miR-182-5p/CYP1B1 axis and highlights circPPAP2B as a potential therapeutic target for ccRCC intervention.
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Carcinoma de Células Renales , Neoplasias Renales , MicroARNs , Humanos , Carcinoma de Células Renales/genética , Empalme Alternativo , ARN Circular/genética , MicroARNs/genética , Neoplasias Renales/genética , Ribonucleoproteínas Nucleares Heterogéneas , Proteína de Unión al Tracto de Polipirimidina , Citocromo P-450 CYP1B1 , Ribonucleoproteína Heterogénea-Nuclear Grupo C/genéticaRESUMEN
Metabolic dysfunction-associated steatotic liver disease (MASLD) is caused by lipid accumulation within the liver. The pathogenesis underlying its development is poorly understood. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon and a group 1 carcinogen. The aryl hydrocarbon receptor activation by B[a]P induces cytochrome P450 (CYP) enzymes, contributing to hepatic lipid accumulation. However, the molecular mechanism through which the B[a]P-mediated induction of CYP enzymes causes hepatic lipid accumulation is unknown. This research was conducted to elucidate the role of CYP1B1 in regulating B[a]P-induced lipid accumulation within hepatocytes. B[a]P increased hepatic lipid accumulation, which was mitigated by CYP1B1 knockdown. An increase in the mammalian target of rapamycin (mTOR) by B[a]P was specifically reduced by CYP1B1 knockdown. The reduction of mTOR increased the expression of autophagic flux-related genes and promoted phagolysosome formation. Both the expression and translocation of TFE3, a central regulator of lipophagy, were induced, along with the expression of lipophagy-related genes. Conversely, enhanced mTOR activity reduced TFE3 expression and translocation, which reduced the expression of lipophagy-related genes, diminished phagolysosome production, and increased lipid accumulation. Our results indicate that B[a]P-induced hepatic lipid accumulation is caused by CYP1B1-induced mTOR and the reduction of lipophagy, thereby introducing novel targets and mechanisms to provide insights for understanding B[a]P-induced MASLD.
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Benzo(a)pireno , Hígado , Benzo(a)pireno/toxicidad , Citocromo P-450 CYP1B1/genética , Hígado/metabolismo , Sistema Enzimático del Citocromo P-450 , Serina-Treonina Quinasas TOR/genética , Receptores de Hidrocarburo de Aril/metabolismo , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Lípidos , Citocromo P-450 CYP1A1/genéticaRESUMEN
Dihydrotanshinone I (DHTI) is a pharmacologically active component occurring in the roots of the herbal medicine Salvia miltiorrhiza Bunge. This study investigated DHTI-induced inhibition of CYP1A1, CYP1A2, and CYP1B1 with the aim to determine the potential effects of DHTI on the bioactivation of estradiol (E2), possibly related to preventive/therapeutic strategy for E2-associated breast cancer. Ethoxyresorufin as a specific substrate for CYP1s was incubated with human recombinant CYP1A1, CYP1A2, or CYP1B1 in the presence of DHTI at various concentrations. Enzymatic inhibition and kinetic behaviors were examined by monitoring the formation of the corresponding product. Molecular docking was further conducted to define the interactions between DHTI and the three CYP1s. The same method and procedure were employed to examine the DHTI-induced alteration of E2 metabolism. DHTI showed significant inhibition of ethoxyresorufin O-deethylation activity catalyzed by CYP1A1, CYP1A2 and CYP1B1 in a concentration-dependent manner (IC50 = 0.56, 0.44, and 0.11 µM, respectively). Kinetic analysis showed that DHTI acted as a competitive type of inhibitor of CYP1A1 and CYP1B1, whereas it noncompetitively inhibited CYP1A2. The observed enzyme inhibition was independent of NADPH and time. Molecular docking analysis revealed hydrogen bonding interactions between DHTI and Asp-326 of CYP1B1. Moreover, DHTI displayed preferential activity to inhibit 4-hydroxylation of E2 (a genotoxic pathway) mediated by CYP1B1. Exposure to DHTI could reduce the risk of genotoxicity induced by E2. SIGNIFICANCE STATEMENT: CYP1A1, CYP1A2, and CYP1B1 enzymes are involved in the conversion of estradiol (E2) into 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) through oxidation. 2-OHE2 is negatively correlated with breast cancer risk, and 4-OHE2 may be a significant initiator and promoter of breast cancer. The present study revealed that dihydrotanshinone I (DHTI) competitively inhibits CYP1A1/CYP1B1 and noncompetitively inhibits CYP1A2. DHTI exhibits a preference for inhibiting the genotoxicity associated with E2 4-hydroxylation pathway mediated by CYP1B1, potentially reducing the risk of 4-OHE2-induced genotoxicity.
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Neoplasias de la Mama , Citocromo P-450 CYP1A2 , Furanos , Fenantrenos , Quinonas , Humanos , Femenino , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Simulación del Acoplamiento Molecular , Cinética , Citocromo P-450 CYP1B1/metabolismo , Estradiol/farmacología , Estradiol/metabolismoRESUMEN
SUMMARY: Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in various types of cancers including breast cancer. However, the role of AhR with its endogenous ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the progression of breast cancer remains poorly understood. We aimed to investigate cell proliferation and migration states in breast cancer after activating AhR with the endogenous ligand ITE. Breast cancer tissue was evaluated by cell lines, immunohistochemistry, reverse transcription-polymerase chain reaction, cell proliferation, flow cytometry, migration assays and western blot techniques. We found that AhR was widely expressed in breast cancer tissues and metastasis lymph node tissues, but not in normal tissues. The expression AhR was independent between the age, grades and TNM classifications for breast cancer tissues. ITE treatment significantly induced the activation of AhR in a time-dependent manner in both MCF-7 and T47D breast cancer cell lines. Meanwhile, ITE did not affect the cell migration but significantly suppressed the cell proliferation in estrogen receptor positive (ER+) MCF-7 andT47D cells, which probably attribute to the induction of cell cycle arrest in G1 phase and shortened S phase. Further mechanism study showed that ERK1/2 and AKT signaling were required for the activation of AhR in MCF-7 cells. These data suggest that AhR is a potential new target for treating patients with breast cancer. ITE may be more potentially used for therapeutic intervention for breast cancer with the kind of ER(+).
El receptor de hidrocarburo de arilo (AhR) es un factor de transcripción activado por ligando que se expresa en gran medida en varios tipos de cáncer, incluido el cáncer de mama. Sin embargo, el papel de AhR con su ligando endógeno 2- (1'H-indol-3'-carbonil)-tiazol-4-ácido carboxílico metil éster (ITE) en la progresión del cáncer de mama sigue siendo poco conocido. Nuestro objetivo fue investigar la proliferación celular y los estados de migración en el cáncer de mama después de activar AhR con el ligando endógeno ITE. El tejido de cáncer de mama se evaluó mediante líneas celulares, inmunohistoquímica, reacción en cadena de la polimerasa con transcriptasa inversa, proliferación celular, citometría de flujo, ensayos de migración y técnicas de transferencia Western. Descubrimos que AhR se expresó ampliamente en tejidos de cáncer de mama y en linfonodos con metástasis, pero no en tejidos normales. La expresión AhR fue independiente entre la edad, grados y clasificaciones TNM para tejidos de cáncer de mama. El tratamiento con ITE indujo significativamente la activación de AhR de manera dependiente del tiempo en las líneas celulares de cancer de mama MCF-7 y T47D. Mientras tanto, ITE no afectó la migración celular, pero suprimió significativamente la proliferación celular en células MCF-7 y T47D con receptor de estrógeno positivo (ER+), lo que probablemente se atribuye a la inducción de la detención del ciclo celular en la fase G1 y la fase S acortada. Un estudio adicional del mecanismo mostró que las señales de ERK1/2 y AKT eran necesarias para la activación de AhR en las células MCF-7. Estos datos sugieren que AhR es un nuevo objetivo potencial para el tratamiento de pacientes con cáncer de mama. ITE puede ser utilizado más potencialmente en la intervención terapéutica para el cáncer de mama con el tipo de ER (+).