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.
UNASSIGNED: 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.

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).
CONCLUSIONS: Variation in the genes identified in this population-based study may help to further explain the genetics of PCG.

Associations between indoor air pollution from fine particulate matter (PM with aerodynamic diameter dp < 2.5 μm) and human health are poorly understood. Here, we analyse the concentration-response curves for fine and ultrafine PM, the gene expression, and the methylation patterns in human bronchial epithelial cells (BEAS-2B) exposed at the air-liquid interface (ALI) within a classroom in downtown Rome. Our results document the upregulation of aryl hydrocarbon receptor (AhR) and genes associated with xenobiotic metabolism (CYP1A1 and CYP1B1) in response to single exposure of cells to fresh urban aerosols at low fine PM mass concentrations within the classroom. This is evidenced by concentrations of ultrafine particles (UFPs, dp < 0.1 μm), polycyclic aromatic hydrocarbons (PAH), and ratios of black carbon (BC) to organic aerosol (OA). Additionally, an interleukin 18 (IL-18) down-regulation was found during periods of high human occupancy. Despite the observed gene expression dysregulation, no changes were detected in the methylation levels of the promoter regions of these genes, indicating that the altered gene expression is not linked to changes in DNA methylation and suggesting the involvement of another epigenetic mechanism in the gene regulation. Gene expression changes at low exposure doses have been previously reported. Here, we add the possibility that lung epithelial cells, when singly exposed to real environmental concentrations of fine PM that translate into ultra-low doses of treatment, may undergo epigenetic alteration in the expression of genes related to xenobiotic metabolism. Our findings provide a perspective for future indoor air quality regulations. We underscore the potential role of indoor UFPs as carriers of toxic molecules with low-pressure weather conditions, when rainfall and strong winds may favour low levels of fine PM.

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.

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 (β/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.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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 34 254 age- and ancestry-matched control individuals, the mean (SD) age was 65.0 (8.4) years, and previous OCP use was reported in 31 177 (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).
UNASSIGNED: 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.

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.

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.

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.

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.