Unveiling compositional images of specific proteins in individual cells by LA-ICP-MS: Labelling with ruthenium red and metal nanoclusters.

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.

Modulation of Angiotensin II-Induced Cellular Hypertrophy by Cannflavin-C: Unveiling the Impact on Cytochrome P450 1B1 and Arachidonic Acid Metabolites.

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.

Cytochrome P450 1B1 is critical in the development of TNF-α, IL-6, and LPS-induced cellular hypertrophy.

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.

Design, synthesis and structure-activity relationship of 1,8-naphthalimide derivatives as highly potent hCYP1B1 inhibitors.

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-Br⋯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.

Gene-Environment Interaction Between CYP1B1 and Oral Contraception on Frontal Fibrosing Alopecia.

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 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). 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.

Exposure of cinnamyl alcohol in co-culture of BEAS-2B and dendritic cells: Interaction between CYP450 and cytokines.

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.

Development of novel N-aryl-2,4-bithiazole-2-amine-based CYP1B1 degraders for reversing drug resistance.

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.

A tryptophan-derived uremic metabolite/Ahr/Pdk4 axis governs skeletal muscle mitochondrial energetics in chronic kidney disease.

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.

CYP1B1 affects the integrity of the blood-brain barrier and oxidative stress in the striatum: An investigation of manganese-induced neurotoxicity.

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.

Emissions from plastic incineration induce inflammation, oxidative stress, and impaired bioenergetics in primary human respiratory epithelial cells.

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.

Dihydrotanshinone I-Induced CYP1 Enzyme Inhibition and Alteration of Estradiol Metabolism.

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.