P450 1A1 Research Articles

A pyrazolopyridine as a novel AhR signaling activator with anti-breast cancer properties in vitro and in vivo

Breast cancer is one of the main causes of malignancy-related deaths globally and has a significant impact on women's quality of life. Despite significant therapeutic advances, there is a medical need for targeted therapies in breast cancer. Aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor mediates responses to environment stimuli, is emerging as a unique pleiotropic target. Herein, a combined molecular simulation and in vitro investigations identified 3-(3-fluorophenyl)-1H-pyrazolo[3,4-b]pyridine (3FPP) as a novel AhR ligand in T47D and MDA-MB-231 breast cancer cells. Its agonistic effects induced formation of the AhR-AhR nuclear translocator (Arnt) heterodimer and prompted its binding to the penta-nucleotide sequence, called xenobiotic-responsive element (XRE) motif. Moreover, 3FPP augmented the promoter-driven luciferase activities and expression of AhR-regulated genes encoding cytochrome P450 1A1 (CYP1A1) and microRNA (miR)-212/132 cluster. It reduced cell viability, migration, and invasion of both cell lines through AhR signaling. These anticancer properties were concomitant with reduced levels of B-cell lymphoma 2 (BCL-2), SRY-related HMG-box4 (SOX4), snail family zinc finger 2 (SNAI2), and cadherin 2 (CDH2). In vivo, 3FPP suppressed tumor growth and activated AhR signaling in an orthotopic mouse model. In conclusion, our results introduce the fused pyrazolopyridine 3FPP as a novel AhR agonist with AhR-specific anti-breast cancer potential in vitro and in vivo.

Circ0087385 promotes DNA damage in benzo(a)pyrene-induced lung cancer development by upregulating CYP1A1.

Increasing environmental genotoxic chemicals have been shown to induce epigenetic alterations. However, the interaction between genetics and epigenetics in chemical carcinogenesis is still not fully understood. Here, we constructed an in vitro human lung carcinogenesis model (16HBE-T) by treating human bronchial epithelial cells with a typical significant carcinogen benzo(a)pyrene (BaP). We identified a novel circular RNA, circ0087385, which was overexpressed in 16HBE-T and human lung cancer cell lines, as well as in lung cancer tissues and serum exosomes from lung cancer patients. The upregulated circ0087385 after exposure to BaP promoted DNA damage in the early stage of chemical carcinogenesis and affected the cell cycle, proliferation, and apoptosis of the malignantly transformed cells. Overexpression of circ0087385 enhanced the expression of cytochrome P450 1A1 (CYP1A1), which is crucial for metabolically activating BaP. Interfering with circ0087385 or CYP1A1 reduced the levels of ultimate carcinogen benzo(a)pyrene diol epoxide (BPDE) and BPDE-DNA adducts. Interfering with CYP1A1 partially reversed the DNA damage induced by high expression of circ0087385, as well as decreased the level of BPDE and BPDE-DNA adducts. These findings provide novel insights into the interaction between epigenetics and genetics in chemical carcinogenesis which are crucial for understanding the epigenetic and genetic toxicity of chemicals.

Pyridinyl 4-(2-oxoalkylimidazolidin-1-yl)benzenesulfonates and their hydrochloride salts as novel water soluble antimitotic prodrugs bioactivated by cytochrome P450 1A1 in breast cancer cells.

We developed first-in-class antimitotic prodrugs phenyl 4-(2-oxo-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) bioactivated by cytochrome P450 (CYP) 1A1 that are highly selective toward several breast cancer cells. However, they show sparingly water solubility. Therefore, we replaced their phenyl ring B with a substituted pyridinyl group preparing novel pyridinyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PYRAIB-SOs) and their hydrochloride salts. Our results evidence that PYRAIB-SO hydrochloride salts show higher water solubility compared to their neutral and PAIB-SO counterparts by up to 625-fold. PYRAIB-SOs with a nitrogen atom at position 3 of the pyridinyl ring exhibited strong antiproliferative activity (IC50: 0.03-3.3 μM) and high selectivity (8->1250) toward sensitive CYP1A1-positive breast cancer cells and cells stably transfected with CYP1A1. They induce cell cycle arrest in the G2/M phase and disrupt microtubule dynamic assembly. Enzymatic assays confirmed that CYP1A1 metabolizes PYRAIB-SOs into their active form with in vitro hepatic half-lives (55-120 min) in rodent and human liver microsomes. Overall, this will allow to increase drug concentration for in vivo studies.

Effect of lipoic acid on the expression of nonalcoholic fatty liver disease-associated genes in the liver of rats fed a hypercaloric choline-deficient diet

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver pathologies. Molecular mechanisms of NAFLD pathogenesis are complex and still require further clarification. Thus, this study aimed to investigate the impact of lipoic acid (LA), widely used as an antioxidant, on the development of different links of NAFLD, including de novo lipogenesis, antioxidant and xenobiotic-metabolizing enzymes by gene expression analysis. The experiment was carried out on three groups of male Wistar rats received control diet, hypercaloric choline-deficient diet (HCCD) or HCCD with LA (HCCD+LA). In the liver of rats the expression of acetyl-CoA carboxylase 1 (Acaca), fatty acid synthase (Fasn), stearoyl-CoA desaturase (Scd) and transcriptional regulators sterol-regulatory element-binding protein SREBP (Srebf1), carbohydrate response element-binding protein ChREBP (Mlxipl), peroxisome proliferator-activated receptor alpha PPARα (Ppara), heme oxygenase-1 HO-1 (Hmox1) and nuclear erythroid 2-related factor 2 Nrf2 (Nrf2), cytochrome P450 1A1 CYP1A1 (CYP1A1) and aryl hydrocarbon receptor AhR (AhR), cytochrome P450 2E1 CYP2E1 (CYP2E1) genes were evaluated. Supplementation of HCCD with LA led to an even greater than in HCCD group decrease in Scd gene expression by 88% (p<0.05), as well as a marked suppression of Ppara and Mlxipl by 37% (p<0.05) and 27% (p<0.05), to an 80% (p<0.05) elevation of Hmox1 gene expression relative to the HCCD group, had no pronounced effect on CYP1A1 and AhR gene expression but resulted in a 38% (p<0.05) suppression of CYP2E1 expression compared with that slightly elevated by HCCD. In conclusion, the current study showed that LA reduces lipogenesis de novo, restores the expression of Hmox1 gene of the antioxidant enzyme HO-1, diminished by the HCCD diet, and decreases HCCD-induced level of the expression of CYP2E1 gene of CYP2E1 enzyme, a potential source of free radicals.

Dietary aflatoxin B1 and zearalenone contamination affected growth performance, intestinal and hepatopancreas gene expression profiles and histology of the intestine and gill in goldfish, Carassius auratus

The effects of dietary aflatoxin B1 (AFB1) and/or zearalenone (ZEA) contamination on goldfish, was investigated. Total of 540 fingerlings, 6.25 ± 0.12 g, were randomly allocated to experimental groups comprised of factorial designation of different dietary AFB1 (0, 50 and 100 ppb) and ZEA (0, 500 and 1000 ppb) concentrations for 60 days. Each treatment was conducted in triplicate. Results showed dietary AFB1 and/or ZEA depressed the growth performance of fish; the highest and lowest weight gain (WG), final body weight, thermal growth coefficient (TGC) and daily growth coefficient (DGC) were observed in control group and AFB50ZEA1000, respectively (P < 0.05). Moreover, dietary AFB1 and/or ZEA significantly affected the gene expressions of Caspase 3, Cytochrome P450 (CYP) 1A1, Glutathione S-transferases (GST) and Heat shock proteins 70 (HSP70) in a dose-dependent and tissue specific manner (hepatopancreas or intestine) (P < 0.05). In the hepatopancreas, the highest relative expression of Caspase 3 (2.08 ± 0.43), CYP1A1 (2.73 ± 1.18), GST (32.86 ± 4.03) and HSP70 (7.55 ± 2.35) genes were observed in AFB100ZEA1000, AFB50ZEA0, AFB0ZEA1000 and AFB0ZEA1000 groups, respectively (P < 0.05). However, the highest relative expression of Caspase 3 (39.70 ± 10.98), CYP1A1 (966.4 ± 68.6), GST (175.8 ± 37.1) and HSP70 (1.01 ± 0.22) genes in the intestinal tissue were observed in AFB100ZEA1000, AFB0ZEA1000, AFB100ZEA500 AFB0ZEA0 groups (P < 0.05). The highest scores of the intestine tissue inflammation (4.0 ± 0.75) and necrosis (4.0 ± 0.75) were observed in AFB100ZEA500 group (P < 0.05). The results indicated the significant contribution of the intestine in metabolism/excretion of the feed-born toxins. Furthermore, the results revealed that dietary AFB1 and/or ZEA contamination could negatively affect fish performance and detoxifying genes expression profiles at concentrations even lower than those indicated in the literature as the safe feed mycotoxin thresholds.

Prenatal exposure to polycyclic aromatic hydrocarbons (PAHs) augments neonatal hyperoxic lung injury: Role of cytochrome P450 (CYP)1A1, 1A2, and 1B1

Pregnant women exposed to polycyclic aromatic hydrocarbons (PAHs) are at increased risk for premature delivery. Premature infants often require supplemental oxygen, a known risk factor for bronchopulmonary dysplasia (BPD). Cytochrome P450 (CYP) enzymes have been implicated in hyperoxic lung injury. We hypothesize that prenatal PAH exposure exacerbates oxygen-mediated lung injury in neonatal mice, and that this effect is differentially altered in mice lacking the gene for (Cyp)1a1, 1a2, or 1b1. Timed pregnant wild type (WT) (C57BL/6J) mice were orally administered a PAH mixture of benzo[a]pyrene (BP) and benzo[b]fluoranthene (BbF) or the vehicle corn oil (CO) once daily on gestational days 16–19, and the dose response on postnatal lung injury was examined. In addition, timed pregnant mice with one of four genotypes, WT, Cyp1a1-null, Cyp1a2-null, and Cyp1b1-null, were treated orally with CO or PAH on gestational days 16–19 and exposed to hyperoxia or room air for 14 days. Lung injury was assessed on PND15 by radial alveolar count (RAC) and mean linear intercept (MLI) Gene expression of DNA repair genes in lung and liver were measured. Results showed that neonatal hyperoxic lung injury is augmented by prenatal PAH exposure in a dose-dependent manner. This effect was differentially altered in the Cyp-null mice, with Cyp1a2-null showing the greatest extent of lung injury. We concluded that newborn mice exposed to PAH in utero had more significant lung injury in response to hyperoxia than non-PAH exposed pups, and that CYP1A1 and CYP1A2 are protective against lung injury while CYP1B1 augments lung injury.

Selective cascade activation of polycyclic aromatic hydrocarbons in human cells: Role of enzyme's intrinsic electric field

Polycyclic aromatic hydrocarbons (PAHs) are major environmental organic pollutants. Some metabolites of PAHs show greater toxicity to humans while the others do not. It is highly important to decipher PAHs' regioselective activation mechanism and identify the major metabolites to accurately evaluate their public health risk. Here, we have performed a thorough computational study of benzo[a]anthracene (BA) metabolized by P450 1A1 by employing molecular docking, molecular dynamics simulations, quantum chemical calculation, and quantum mechanics/molecular mechanics calculations. Our findings show that highly-reactive species such as 3,4-epoxide, 8,9-epoxide, 3,4-diol-1,2-epoxide, and 8,9-diol-10,11-epoxide were major metabolites, which can efficiently react with guanine and damage DNA with extremely low energy barrier, therefore, supports the regioselective metabolism of BA. The origin of this selective activation is mainly contributed to both the oxygen‑carbon distance and previously overlooked enzyme's intrinsic electric field. Consequently, based on the resolved activation selectivity of BA. We built a high-throughput strategy to efficiently predict the metabolites of other PAHs. The accuracy of the strategy is validated by studying 16 PAHs on the priority control list. Hopefully this will aid the accurate evaluation of public health risks associated with PAH emissions.

Etoricoxib enhances aryl hydrocarbon receptor activity

Etoricoxib is a nonsteroidal anti-inflammatory drug (NSAID) that possesses properties that include reducing inflammation and relieving pain and fever. Etoricoxib is an oral medication that selectively inhibits cyclooxygenase-2 with high efficacy. Controversies about its cardiovascular side effects have long existed. The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor that plays a key role in the metabolism of xenobiotics and many physiological functions. 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is a tryptophan metabolite and endogenous AhR agonist. Activation of AhR by its ligand induces upregulation of AhR-targeted cytochrome P450 (CYP) 1A1 expression. We found that etoricoxib (10–60 μM) induced CYP1A1 mRNA and protein expressions and the transcriptional activity of AhR mediated by the aryl hydrocarbon response element (AHRE) in both mouse Hepa-1c1c7 and human HepG2 cells. Its induction did not appear in AhR signaling-deficient cells, and was inhibited by the AhR antagonist, CH-223191. Etoricoxib was able to induced the translocalization of AhR from cytosol into nucleus. Etoricoxib also worked synergistically with ITE to further increase the expression of CYP1A1 mRNA and protein in human cells. The synergistic effect was higher in cells with than cells without overexpression of AhR. In summary, etoricoxib is an agonist of AhR in both mouse and human cells. Etoricoxib has a synergistic effect on ITE-induced CYP1A1 expression in human cells. The effect of etoricoxib on AhR and ITE on endothelial cells and cardiomyocytes should be further elucidated to in hope to clarify the mechanism of increased cardiovascular events in COX-2 inhibitors and etoricoxib.

CYP1A1 immunohistochemistry is highly specific for angiofibroma of soft tissue among morphological mimics.

Angiofibroma of soft tissue (AFST) is a benign, morphologically distinctive tumour type that harbours recurrent AHRR::NCOA2 fusions in 60-70% of cases and shows a non-specific immunophenotype, expressing EMA in roughly half of cases. The AHRR::NCOA2 fusion results in increased expression of cytochrome P450 1A1 (CYP1A1); a recent study demonstrated CYP1A1 immunohistochemistry (IHC) to be moderately sensitive and highly specific for AFST. In this study, we sought to validate these findings in a larger independent cohort of 30 AFST, as well as 215 morphological mimics, including 30 solitary fibrous tumours, 29 myxoid liposarcomas, 28 low-to-intermediate grade myxofibrosarcomas (MFS), 20 atypical spindle cell lipomatous tumours (ASCLT), 20 cellular angiofibromas, 10 cases each of spindle cell lipoma, neurofibroma, malignant peripheral nerve sheath tumour, superficial angiomyxoma, cellular myxoma, soft tissue perineurioma and deep fibrous histiocytoma, and nine cases each of low-grade fibromyxoid sarcoma and mammary-type myofibroblastoma. We found CYP1A1 IHC to be 70% sensitive for AFST, with granular cytoplasmic staining in 21 of 30 tumours, and 98% specific, with staining in only five morphological mimics: two deep fibrous histiocytomas, one MFS, one cellular angiofibroma and one ASCLT. These findings confirm that CYP1A1 is 70% sensitive, consistent with the prevalence of AHRR::NCOA2 fusions that up-regulate this protein, and that it is highly specific among morphological mimics.

Mulberry leaf supplementation inhibits skatole deposition by regulating gut microbiota and upregulating liver cytochrome P450 1A1 expression in finishing pigs

Skatole, a strong fecal odor substance, is generated through microbial degradation of tryptophan in the animal hindgut. It easily accumulates in adipose tissue and affects meat quality. In this study, the effect of mulberry leaf supplementation on skatole in finishing pigs was studied. In a 35-day trial, 20 finishing pigs (barrows and gilts) were fed with a basal diet or basal diet with 6% mulberry leaves. Growth performance of the pigs (n = 10) was automatically recorded by a performance-testing feeder system and 8 pigs in each treatment were slaughtered and sampled for the remaining tests. Skatole and short-chain fatty acids were detected using HPLC and gas chromatography, respectively. Fecal microbiota were analyzed using 16S rRNA gene sequencing. The metabolomics analysis of feces and serum was performed with UHPLC-MS/MS. The major cytochrome P450 (CYP) enzymes that catalyze skatole degradation in the liver were tested by using RT-PCR and Western blot. Effects of major bioactive compounds in mulberry leaves on the CYP genes were verified in the hepatic cell line HepG2 in an in vitro test (n = 3). In finishing pigs, mulberry leaf supplementation had no significant effect on the average daily gain, average daily feed intake, and feed conversion ratio (P > 0.05), but reduced skatole levels in feces, serum, and backfat (P < 0.05), and increased acetic acid levels in feces (P = 0.027). Mulberry leaf supplementation decreased the relative abundance of the skatole-producing bacteria Megasphaera and Olsenella (P < 0.05). Indole-3-acetic acid, the intermediate that is essential for skatole production, was significantly reduced in feces by mulberry leaf supplementation (P < 0.05) and was positively correlated with skatole content in feces (P = 0.004). In pigs treated with mulberry leaves, liver CYP1A1 expression was increased (P < 0.05) and was negatively correlated with skatole content in backfat (P = 0.045). The in vitro test demonstrated that mulberry leaf polyphenols and polysaccharides could directly stimulate CYP1A1 expression in hepatic cells. These findings suggest that mulberry leaf supplementation reduces skatole production and deposition in finishing pigs by regulating the gut microbiota and promoting skatole degradation in liver.

Melatonin Activation by Human Cytochrome P450 Enzymes: A Comparison between Different Isozymes.

Cytochrome P450 enzymes in the human body play a pivotal role in both the biosynthesis and the degradation of the hormone melatonin. Melatonin plays a key role in circadian rhythms in the body, but its concentration is also linked to mood fluctuations as well as emotional well-being. In the present study, we present a computational analysis of the binding and activation of melatonin by various P450 isozymes that are known to yield different products and product distributions. In particular, the P450 isozymes 1A1, 1A2, and 1B1 generally react with melatonin to provide dominant aromatic hydroxylation at the C6-position, whereas the P450 2C19 isozyme mostly provides O-demethylation products. To gain insight into the origin of these product distributions of the P450 isozymes, we performed a comprehensive computational study of P450 2C19 isozymes and compared our work with previous studies on alternative isozymes. The work covers molecular mechanics, molecular dynamics and quantum mechanics approaches. Our work highlights major differences in the size and shape of the substrate binding pocket amongst the different P450 isozymes. Consequently, substrate binding and positioning in the active site varies substantially within the P450 isozymes. Thus, in P450 2C19, the substrate is oriented with its methoxy group pointing towards the heme, and therefore reacts favorably through hydrogen atom abstraction, leading to the production of O-demethylation products. On the other hand, the substrate-binding pockets in P450 1A1, 1A2, and 1B1 are tighter, direct the methoxy group away from the heme, and consequently activate an alternative site and lead to aromatic hydroxylation instead.

BPDE-DNA adduct formation and alterations of mRNA, protein, and DNA methylation of CYP1A1, GSTP1, and GSTM1 induced by benzo[a]pyrene and the intervention of aspirin in mice.

Benzo[a]pyrene (B[a]P), one typical environmental pollutant, the toxicity mechanisms, and potential prevention remain perplexing. Available evidence suggests cytochrome P450 1A1 (CYP1A1) and glutathione S-transferases (GSTs) metabolize B[a]P, resulting in metabolic activation and detoxification of B[a]P. This study aimed to reveal the impact of B[a]P exposure on trans-7,8-diol-anti-9,10-epoxide DNA (BPDE-DNA) adduct formation, level of CYP1A1, glutathione S-transferase pi (GSTP1) and glutathione S-transferase mu1 (GSTM1) mRNA, protein and DNA methylation in mice, and the potential prevention of aspirin (ASP). This study firstly determined the BPDE-DNA adduct formation in an acute toxicity test of a large dose in mice induced by B[a]P, which subsequently detected CYP1A1, GSTP1, and GSTM1 at levels of mRNA, protein, and DNA methylation in the organs of mice in a subacute toxicity test at appropriate doses and the potential prevention of ASP, using the methods of real-time quantitative PCR (QPCR), western blotting, and real-time methylation-specific PCR (MSP), respectively. The results verified that B[a]P induced the formation of BPDE-DNA adduct in all the organs of mice in an acute toxicity test, and the order of concentration of which was lung > kidney > liver > brain. In a subacute toxicity test, following B[a]P treatment, mice showed a dose-dependent slowdown in body weight gain and abnormalities in behavioral and cognitive function and which were alleviated by ASP co-treatment. Compared to the controls, following B[a]P treatment, CYP1A1 was significantly induced in all organs in mice at mRNA level (P < 0.05), was suppressed in the lung and cerebrum of mice at protein level, and inhibited at DNA methylation level in the liver, lung, and cerebrum, whereas GSTP1 and GSTM1 at mRNA, protein, and DNA methylation levels showed organ-specific changes in mice following B[a]P treatment, which was generally alleviated by ASP intervention. In conclusion, B[a]P induced BPDE-DNA adduct formation in all organs in mice and altered the mRNA, protein, and DNA methylation levels in CYP1A1, GSTP1, and GSTM1 in an organ-dependent pattern, which could be related to the organ toxicity and mechanism of B[a]P. ASP intervention may be an effective measure to prevent B[a]P toxicity. The findings provide scientific evidence for further study on the organ toxicity and mechanisms of B[a]P.

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamide salts: Novel hydrosoluble prodrugs of antimitotics selectively bioactivated by the cytochrome P450 1A1 in breast cancer cells

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides (PAIB-SAs) are members of a new family of prodrugs bioactivated by cytochrome P450 1A1 (CYP1A1) in breast cancer cells into their potent 4-(2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamide metabolites (PIB-SAs). One of the predominant problems for the galenic formulation and administration of PAIB-SAs in animal studies is their poor hydrosolubility. To circumvent that difficulty, we report the design, the synthesis, the chemical characterization, the evaluation of the aqueous solubility, the antiproliferative activity and the mechanism of action of 18 new Na+, K+ and Li+ salts of PAIB-SAs. Our results evidenced that the latter exhibited highly selective antiproliferative activity toward MCF7 and MDA-MB-468 breast cancer cells expressing endogenously CYP1A1 compared to insensitive MDA-MB-231 and HaCaT cells. Moreover, PAIB-SA salts 1–18 are significantly more hydrosoluble (3.9–9.4 mg/mL) than their neutral counterparts (< 0.0001 mg/mL). In addition, the most potent PAIB-SA salts 1–3 and 10–12 arrested the cell cycle progression in the G2/M phase and disrupted the cytoskeleton’s dynamic assembly. Finally, PAIB-SA salts are N-dealkylated by CYP1A1 into their corresponding PIB-SA metabolites, which are potent antimitotics. In summary, our results show that our water-soluble PAIB-SA salts, notably the sodium salts, still exhibit potent antiproliferative efficacy and remain prone to CYP1A1 bioactivation. In addition, these PAIB-SA salts will allow the development of galenic formulations suitable for further biopharmaceutical and pharmacodynamic studies.

Bojungikgi-tang improves skin barrier function and immune response in atopic dermatitis mice fed a low aryl hydrocarbon receptor ligand diet

BackgroundThe aryl hydrocarbon receptor (AhR) is a transcription factor that plays a crucial role in regulating the immune system and maintaining skin barrier function. AhR signaling is pivotal in the pathogenesis of inflammatory diseases such as atopic dermatitis (AD), and the absence of AhR ligands further contributes to the progression or worsening of AD symptoms.MethodsAD was induced with 2,4-dinitrochlorobenzene (DNCB), and Bojungikgi-tang (BJIKT) was administered orally daily for 10 weeks. Serum IgE, splenocyte IL-4, and IFN-γ levels, skin barrier genes, and AhR target gene expressions were analyzed using RNA-sequencing analysis. Spleen tissues were extracted for fluorescence-activated cell sorting (FACS) analysis to analyze the effect of BJIKT on immune responses. A correlation analysis was conducted to analyze the correlation between immune markers and skin barrier genes and AhR target genes.ResultsBJIKT effectively improved AD symptoms in AD mice fed a low AhR ligand diet by reducing neutrophil and eosinophil counts, lowering IgE levels in the blood, and decreasing IL-4 and IFN-γ levels in the splenocytes. Additionally, BJIKT significantly reduced epithelial skin thickness and transepidermal water loss (TEWL) values and reversed the decreased expression of skin barrier genes. BJIKT also considerably altered the expression of AhR target genes, including Ahr, Ahrr, cytochrome P450 1A1 (CYP1A1), and CYP1B1. Furthermore, AhR target pathway genes were negatively correlated with immune cell subtypes, including CD4 + and CD8 + T cells and macrophages (CD11b + F4/80 +) at the systemic level.ConclusionsBJIKT can regulate AhR activation and may help reduce inflammation in AD by regulating the expression of skin barrier genes and immune responses.

StemRegenin-1 Attenuates Endothelial Progenitor Cell Senescence by Regulating the AhR Pathway-Mediated CYP1A1 and ROS Generation.

Endothelial progenitor cell (EPC)-based stem cell therapy is a promising therapeutic strategy for vascular diseases. However, continuous in vitro expansion for clinical studies induces the loss of EPC functionality due to aging. In this study, we investigated the effects of StemRegenin-1 (SR-1), an antagonist of aryl hydrocarbon receptor (AhR), on replicative senescence in EPCs. We found that SR-1 maintained the expression of EPC surface markers, including stem cell markers, such as CD34, c-Kit, and CXCR4. Moreover, SR-1 long-term-treated EPCs preserved their characteristics. Subsequently, we demonstrated that SR-1 showed that aging phenotypes were reduced through senescence-associated phenotypes, such as β-galactosidase activity, SMP30, p21, p53, and senescence-associated secretory phenotype (SASP). SR-1 treatment also increased the proliferation, migration, and tube-forming capacity of senescent EPCs. SR-1 inhibited the AhR-mediated cytochrome P450 (CYP)1A1 expression, reactive-oxygen species (ROS) production, and DNA damage under oxidative stress conditions in EPCs. Furthermore, as a result of CYP1A1-induced ROS inhibition, it was found that accumulated intracellular ROS were decreased in senescent EPCs. Finally, an in vivo Matrigel plug assay demonstrated drastically enhanced blood vessel formation via SR-1-treated EPCs. In summary, our results suggest that SR-1 contributes to the protection of EPCs against cellular senescence.

A Fluorescent Chemosensor for Long‐Term Tracking of Cancer Cell Metastasis and Invasion via Enzyme‐Activated Anchoring

AbstractEvidence shows that the enzyme human cytochrome P450 1A1 (CYP1A1) is associated with cancer; indeed, it is shown to play a key role in the occurrence of many cancers. Therefore, the molecular imaging of CYP1A1 in cells is of great significance for revealing the process of cancer development. Herein, a chemosensor, DCBEM, is reported that is able to selectively recognize CYP1A1 and achieve long‐term labeling of the enzyme through an enzymolysis cascade reaction. The design of DCBEM relies on the reaction between the highly electrophilic intermediate (quinone methide) and the enzyme, which forms a fluorescent label with CYP1A1 via covalent bonding. The chemosensor reveals excellent specificity toward CYP1A1 and achieves high‐resolution monitoring of cell migration with a strong retention capacity in vivo (up to 4 days). Further, using DCBEM, the pathways of invasion of colon cancer and breast cancer cells are successfully visualized in living mice. This method of labeling enzymes provides a simple and efficient way to render ordinary fluorescent chemosensors suitable for the long‐term tracking of cancer cells, for which such molecular tools are currently lacking.

DESIGNER fraction concept unmasks minor bioactive constituents in red clover (Trifolium pratense L.)

In botanical extracts, highly abundant constituents can mask or dilute the effects of other, and often, more relevant biologically active compounds. To facilitate the rational chemical and biological assessment of these natural products with wide usage in human health, we introduced the DESIGNER approach of Depleting and Enriching Selective Ingredients to Generate Normalized Extract Resources. The present study applied this concept to clinical Red Clover Extract (RCE) and combined phytochemical and biological methodology to help rationalize the utility of RCE supplements for symptom management in postmenopausal women. Previous work has demonstrated that RCE reduces estrogen detoxification pathways in breast cancer cells (MCF-7) and, thus, may serve to negatively affect estrogen metabolism-induced chemical carcinogenesis. Clinical RCE contains ca. 30% of biochanin A and formononetin, which potentially mask activities of less abundant compounds. These two isoflavonoids are aryl hydrocarbon receptor (AhR) agonists that activate P450 1A1, responsible for estrogen detoxification, and P450 1B1, producing genotoxic estrogen metabolites in female breast cells. Clinical RCE also contains the potent phytoestrogen, genistein, that downregulates P450 1A1, thereby reducing estrogen detoxification. To identify less abundant bioactive constituents, countercurrent separation (CCS) of a clinical RCE yielded selective lipophilic to hydrophilic metabolites in six enriched DESIGNER fractions (DFs 01–06). Unlike solid-phase chromatography, CCS prevented any potential loss of minor constituents or residual complexity (RC) and enabled the polarity-based enrichment of certain constituents. Systematic analysis of estrogen detoxification pathways (ERα-degradation, AhR activation, CYP1A1/CYP1B1 induction and activity) of the DFs uncovered masked bioactivity of minor/less abundant constituents including irilone. These data will allow the optimization of RCE with respect to estrogen detoxification properties. The DFs revealed distinct biological activities between less abundant bioactives. The present results can inspire future carefully designed extracts with phytochemical profiles that are optimized to increase in estrogen detoxification pathways and, thereby, promote resilience in women with high-risk for breast cancer. The DESIGNER approach helps to establish links between complex chemical makeup, botanical safety and possible efficacy parameters, yields candidate DFs for (pre)clinical studies, and reveals the contribution of minor phytoconstituents to the overall safety and bioactivity of botanicals, such as resilience promoting activities relevant to women's health.

Exploring the anticancer potential of thiadiazole derivatives of substituted thiosemicarbazones formed via copper‐mediated cyclization

A couple of N‐(4)‐morpholine/pyrrolidine‐substituted thiosemicarbazones (TSCs) of fluorene‐2‐carboxaldehyde (FM and FP), and their corresponding thiadiazoles (TDZs) (CFM and CFP), were synthesized and characterized (elemental analysis, ultraviolet–visible [UV–Visible], Fourier transform infrared [FT‐IR], nuclear magnetic resonance [NMR; 1H &amp; 13C], high‐resolution mass spectrometry [HRMS], and single‐crystal X‐ray diffraction [SCXRD]) for the evaluation of their anticancer potential. The TDZs were obtained unexpectedly and are possibly formed via single‐step metal (copper)‐mediated oxidative cyclizations of the TSCs. The synthesized compounds are fairly stable in phosphate buffer at the biological pH of 7.4. The density functional theory [DFT] studies were performed to predict the optimized structures and physicochemical properties of these compounds. The compounds were further subjected to computational and experimental biomolecular investigations in order to evaluate their anticancer activity in detail. CFM had the most potent activity against human breast adenocarcinoma (MCF‐7) and human urinary bladder (T24) cancer cells, with IC50 values of 12.00 and 24.80 μM, respectively. In contrast, CFM had negligible cytotoxicity (IC50 = 98.70 μM) against kidney epithelial cells extracted from an African green monkey (Vero) normal cells. This outcome was preferable to that of the widely used medicine Cisplatin. Molecular docking studies were performed with the breast cancer protein “cytochrome P450 1A1” (CYP1A1) and bovine serum albumin (BSA) to predict how effectively the compounds bind to the receptor. The ADMET findings suggest that these compounds have considerable drug‐likeness and oral bioavailability. These insights may open the door for additional medical research into the bioactivities of TSCs and TDZs produced from bioactive carbonyl compounds.

Inhibition of TRPA1, Endoplasmic Reticulum Stress, Human Airway Epithelial Cell Damage, and Ectopic MUC5AC Expression by Vasaka (Adhatoda vasica; Malabar Nut) Tea.

This study tested whether a medicinal plant, Vasaka, typically consumed as a tea to treat respiratory malaise, could protect airway epithelial cells (AECs) from wood smoke particle-induced damage and prevent pathological mucus production. Wood/biomass smoke is a pneumotoxic air pollutant. Mucus normally protects the airways, but excessive production can obstruct airflow and cause respiratory distress. Vasaka tea pre- and co-treatment dose-dependently inhibited mucin 5AC (MUC5AC) mRNA induction by AECs treated with wood smoke particles. This correlated with transient receptor potential ankyrin-1 (TRPA1) inhibition, an attenuation of endoplasmic reticulum (ER) stress, and AEC damage/death. Induction of mRNA for anterior gradient 2, an ER chaperone/disulfide isomerase required for MUC5AC production, and TRP vanilloid-3, a gene that suppresses ER stress and wood smoke particle-induced cell death, was also attenuated. Variable inhibition of TRPA1, ER stress, and MUC5AC mRNA induction was observed using selected chemicals identified in Vasaka tea including vasicine, vasicinone, apigenin, vitexin, isovitexin, isoorientin, 9-oxoODE, and 9,10-EpOME. Apigenin and 9,10-EpOME were the most cytoprotective and mucosuppressive. Cytochrome P450 1A1 (CYP1A1) mRNA was also induced by Vasaka tea and wood smoke particles. Inhibition of CYP1A1 enhanced ER stress and MUC5AC mRNA expression, suggesting a possible role in producing protective oxylipins in stressed cells. The results provide mechanistic insights and support for the purported benefits of Vasaka tea in treating lung inflammatory conditions, raising the possibility of further development as a preventative and/or restorative therapy.

Abstract 73: Specific Activity of GSTs and Protein Expression of GSTT1, GSTP1 and CYP1A1 in Head and Neck Cancer

Abstract Purpose: Rising incidence of head and neck cancer have been observed in developing countries over last decade. It has been postulated that carcinogen detoxifying genes may be involved directly or indirectly in pathogenesis of head and neck cancer. Three genes (CYP1A1, GSTM1 and GSTP1) were studied for their association with head and neck cancer. Cytochrome P450 1A1 (CYP1A1) is phase I enzyme while glutathione S transferases (GSTs) constitute the phase II detoxification enzymes. Abnormal protein expression of CYP1A1 and GSTs lead to cancer initiation. Methods: The current case control study was designed to evaluate the possible associations of these enzymes with head and neck cancer. Serum GSTs activity was determined by using ELISA and CYP1A1, GSTT1, GSTP1 expression in tissue by immunohistochemistry with specific antibodies. Results: Mean serum GSTs specific activity was 7.7 (+11.5)U/L and control was 11.4 (+7.5)U/L. Significant decrease (P&amp;lt;0.05) in GSTs specific activity was observed in head and neck cancer patients compared to controls (P&amp;lt;0.001). Moreover, CYP1A1 and GSTT1 was down expressed whereas GSTP1 was over expressed in head and neck cancer tissues compared with adjacent normal control tissue. Conclusion: Conclusion: Decreased activity of GSTs, reduced expression of GSTs, CYP1A1, GSTT1 and increased expression of GSTP1 may contribute to head and neck cancer development in Pakistani population. Citation Format: Nosheen Masood, Azra Yasmin. Specific Activity of GSTs and Protein Expression of GSTT1, GSTP1 and CYP1A1 in Head and Neck Cancer [abstract]. In: Proceedings of the 11th Annual Symposium on Global Cancer Research; Closing the Research-to-Implementation Gap; 2023 Apr 4-6. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(6_Suppl):Abstract nr 73.