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The lung epithelial barrier serves as a guardian towards environmental insults and responds to allergen encounter with a cascade of immune reactions that can possibly lead to inflammation. Whether the environmental sensor aryl hydrocarbon receptor (AhR) together with its downstream targets cytochrome P450 (CYP1) family members contribute to the regulation of allergic airway inflammation remains unexplored. By employing knockout mice for AhR and for single CYP1 family members, we found that AhR-/- and CYP1B1-/- but not CYP1A1-/- or CYP1A2-/- animals display enhanced allergic airway inflammation compared to WT. Expression analysis, immunofluorescence staining of murine and human lung sections and bone marrow chimeras suggest an important role of CYP1B1 in non-hematopoietic lung epithelial cells to prevent exacerbation of allergic airway inflammation. Transcriptional analysis of murine and human lung epithelial cells indicates a functional link of AhR to barrier protection/inflammatory mediator signaling upon allergen challenge. In contrast, CYP1B1 deficiency leads to enhanced expression and activity of CYP1A1 in lung epithelial cells and to an increased availability of the AhR ligand kynurenic acid following allergen challenge. Thus, differential CYP1 family member expression and signaling via the AhR in epithelial cells represents an immunoregulatory layer protecting the lung from exacerbation of allergic airway inflammation.

Recently we showed that 2-methoxyestradiol (2-ME), an estrogen (E2) metabolite generated by CYP1B1 (cytochrome P450 1B1) in the paraventricular nucleus (PVN), protects female mice from Ang (angiotensin) II-induced hypertension and increased renal sympathetic activity. We also demonstrated that group IV cPLA 2 α (cytosolic phospholipase A 2 α) in the brain contributes to Ang II-induced hypertension in male mice. This study was conducted to determine the contribution of central cPLA 2 α and its relationship to CYP1B1 in Ang II-induced hypertension in female mice. cPLA 2 α knockdown in the PVN by transduction with adenovirus (Ad)-cPLA 2 α-short hairpin (sh)RNA (200 nL, bilaterally, 1.0х10 12 pfu/mL) but not its control Ad-scrambled (Scr)-shRNA (2.5х10 11 pfu/mL) in ovariectomized (OVX) wild-type ( cPLA 2 α +/+ / Cyp1b1 +/+ , n=8/group) and intact cPLA 2 α +/+ / Cyp1b1 -/- (n=12/group) female mice attenuated the effect of Ang II (700 ng/kg/min, subcutaneous, osmotic pump, 2 weeks) to increase the systolic blood pressure (SBP, mmHg) as measured by tail-cuff (Day 12: 129±3 vs 168±7 and 119±3 vs 172±5, respectively, P2 α in the PVN by transduction with Ad-cPLA 2 α-DNA (1.1х10 12 pfu/mL) but not its control Ad-GFP-DNA (1.0х10 11 pfu/mL) in OVX- cPLA 2 α -/- / Cyp1b1 +/+ mice (n=4/group) restored the effect of Ang II to increase SBP (Day 12: 163±7 vs 124±4, P2 α-shRNA but not Ad-Scr-shRNA transduction in the PVN in OVX- cPLA 2 α +/+ / Cyp1b1 +/+ and intact cPLA 2 α +/+ / Cyp1b1 -/- mice reduced and Ad-cPLA 2 α-DNA but not Ad-GFP-DNA transduction in the PVN in OVX- cPLA 2 α -/- / Cyp1b1 +/+ mice restored the effect of Ang II to increase the renal sympathetic activity as indicated by urinary norepinephrine level (ng/mL, 324±36 vs 707±94, 359±49 vs 979±70, 690±44 vs 421±43, respectively, n=4/group, P2 α activity in the PVN. Thus, 2-ME and/or agents inhibiting cPLA 2 α activity could be useful for treating hypertension and its pathogenesis in females.

BackgroundDespite long-standing recognition in the significance of mucus overproduction in asthma, its etiology remains poorly understood. Muc5ac is a secretory mucin that has been associated with reduced pulmonary function and asthma exacerbations.ObjectivesWe sought to investigate the immunological pathway that controls Muc5ac expression and allergic airway inflammation in asthma.MethodsCockroach allergen-induced Muc5ac expression and aryl hydrocarbon receptor (AhR) signaling activation was examined in the human bronchial epithelial cells (HBECs) and mouse model of asthma. AhR regulation of Muc5ac expression, mitochondrial ROS (Mito-ROS) generation, and NLRP3 inflammasome was determined by AhR knockdown, the antagonist CH223191, and AhR-/- mice. The role of NLRP3 inflammasome in Muc5ac expression and airway inflammation was also investigated.ResultsCockroach allergen induced Muc5ac overexpression in HBECs and airways of asthma mouse model. Increased expression of AhR and its downstream genes CYP1A1 and CYP1B1 was also observed. Mice with AhR deletion showed increased allergic airway inflammation and MUC5AC expression. Moreover, cockroach allergen induced epithelial NLRP3 inflammasome activation (e.g., NLRP3, Caspase-1, and IL-1β), which was enhanced by AhR knockdown or the antagonist CH223191. Furthermore, AhR deletion in HBECs led to enhanced ROS generation, particularly Mito-ROS, and inhibition of ROS or Mito-ROS subsequently suppressed the inflammasome activation. Importantly, inhibition of the inflammasome with MCC950, a NLRP3-specifc inhibitor, attenuated allergic airway inflammation and Muc5ac expression. IL-1β generated by the activated inflammasomes mediated cockroach allergen-induced Muc5ac expression in HBECs.ConclusionsThese results reveal a previously unidentified functional axis of AhR-ROS-NLRP3 inflammasome in regulating Muc5ac expression and airway inflammation.

Background Bupi Yishen Formula (BYF), a patent traditional Chinese medicine (TCM) formulation, has been used in the clinical treatment of chronic kidney disease (CKD). However, the mechanism of action of BYF has not been fully elucidated. Method To investigate the variation in the metabolic profile in response to BYF treatment in a rat model of 5/6 nephrectomy (Nx), rats in the treatment groups received low- or high-dose BYF. At the end of the study, serum and kidney samples were collected for biochemical, pathological, and western blotting analysis. Metabolic changes in serum were analyzed by liquid chromatography-tandem mass spectrometry. Results The results showed that BYF treatment could reduce kidney injury, inhibit inflammation and improve renal function in a dose-dependent manner. In total, 405 and 195 metabolites were identified in negative and positive ion modes, respectively. Metabolic pathway enrichment analysis of differential metabolites based on the Kyoto Encyclopedia of Genes and Genomes database identified 35 metabolic pathways, 3 of which were related to tryptophan metabolism. High-dose BYF reduced the level of kynurenic acid (KA) by more than 50%, while increasing melatonin 25-fold and indole-3-acetic acid twofold. Expression levels of aryl hydrocarbon receptor (AhR), Cyp1A1, and CyP1B1 were significantly reduced in the kidney tissue of rats with high-dose BYF, compared to 5/6 Nx rats. Conclusion BYF has a reno-protective effect against 5/6 Nx-induced CKD, which may be mediated via inhibition of the tryptophan-KA-AhR pathway.

Benzo[a]pyrene (B[a]P) is a potentially hepatotoxic group-1 carcinogen taken up by the body through ingestion of daily foods. B[a]P is widely known to cause DNA and protein damages, which are closely related to cell transformation. Accordingly, studies on natural bioactive compounds that attenuate such chemical-induced toxicities have significant impacts on public health. This study aimed to uncover the mechanism of curcumin, the major curcuminoid in turmeric (Curcuma longa), in modulating the lipid accumulation and oxidative stress mediated by B[a]P cytotoxicity in HepG2 cells. Curcumin treatment reduced the B[a]P-induced lipid accumulation and reactive oxygen spicies (ROS) upregulation and recovered the cell viability. Cytochrome P450 family 1 subfamily A polypeptide 1 (CYP1A1) and Cytochrome P450 subfamily B polypeptide 1 (CYP1B1) downregulation resulting from decreased aryl hydrocarbon receptor (AhR) translocation into nuclei attenuated the effects of B[a]P-induced lipid accumulation and repressed cell viability, respectively. Moreover, the curcumin-induced reduction in ROS generation decreased the nuclear translocation of Nuclear factor erythroid-2-related factor 2 (Nrf2) and the expression of phase-II detoxifying enzymes. These results indicate that curcumin suppresses B[a]P-induced lipid accumulation and ROS generation which can potentially induce nonalcoholic fatty liver disease (NAFLD) and can shed a light on the detoxifying effect of curcumin.

Simple Summary Analysis of the eye liquids collected from a cohort of primary open-angle glaucoma patients identified signaling lipids, the pattern of which suggests a role of arachidonic acid/platelet activating-factor (PAF)-dependent pathways and oxidative stress in the pathogenesis of the disease and provides novel targets for its diagnostics and treatment. Abstract Primary open-angle glaucoma (POAG) is characterized by degeneration of retinal ganglion cells associated with an increase in intraocular pressure (IOP) due to hindered aqueous humor (AH) drainage through the trabecular meshwork and uveoscleral pathway. Polyunsaturated fatty acids and oxylipins are signaling lipids regulating neuroinflammation, neuronal survival and AH outflow. Among them, prostaglandins have been previously implicated in glaucoma and employed for its treatment. This study addressed the role of signaling lipids in glaucoma by determining their changes in AH accompanying IOP growth and progression of the disease. Eye liquids were collected from patients with POAG of different stages and cataract patients without glaucoma. Lipids were identified and quantified by UPLC-MS/MS. The compounds discriminating glaucoma groups were recognized using ANCOVA and PLS-DA statistic approaches and their biosynthetic pathways were predicted by bioinformatics. Among 22 signaling lipids identified in AH, stage/IOP-dependent alterations in glaucoma were provided by a small set of mediators, including 12,13-DiHOME, 9- and 13-HODE/KODE, arachidonic acid and lyso-PAF. These observations correlated with the expression of cytochromes P450 (CYPs) and phospholipases A2 in the ocular tissues. Interestingly, tear fluid exhibited similar lipidomic alterations in POAG. Overall, POAG may involve arachidonic acid/PAF-dependent pathways and oxidative stress as evidenced from an increase in its markers, KODEs and 12,13-DiHOME. The latter is a product of CYPs, one of which, CYP1B1, is known as POAG and primary congenital glaucoma-associated gene. These data provide novel targets for glaucoma treatment. Oxylipin content of tear fluid may have diagnostic value in POAG.

Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.

Aims: Primary congenital glaucoma (PCG) is a leading cause of childhood blindness. The cytochrome P450 family 1, subfamily B, polypeptide 1 (CYP1B1) is the most mutated gene that is associated with PCG. Very few studies have examined the promoter region and exon1 of the CYP1B1 gene. This work was planned to contribute to the description of the possible causative mutations of CYP1B1 gene that are related to PCG affected Egyptian patients. Patients and Methods: Patients diagnosed as glaucomatous based on their symptoms and detailed ophthalmological examinations at the time of presentation underwent an intraocular pressure lowering surgical procedure. Investigations were further proceeded on the molecular level. Sequencing-based mutation screen for the promoter region, exon1 and the coding region of exon3 of CYP1B1 gene have been performed in two related consanguineous PCG affected families and four other sporadic Egyptian patients using the polymerase chain reaction (PCR) assay; where PCR products were sequenced, and further analyzed. Results: Sequencing analysis revealed three novel mutations in PCG affected patients one in the promoter region (g.G2872A) and two in exon1 (g.C3268T and g.C3332T). Two additional mutations in exon3 (p.L432V and p.N453S) reported for the first time in PCG affected Egyptian patients. Clinical and genetic data of the two consanguineous families revealed that although the four parents have the same variations as their sons, they are ophthalmologically free. Conclusion: Regular ophthalmic examinations of siblings and parents of these affected patients should take place for early detection of any form of glaucoma to allow prompt diagnosis and early treatment when needed. Clinical examination and molecular genetic data could contribute to early diagnosis and prevention of the visual impairment caused by PCG. This study provides groundwork for expanded genetic investigations in Egypt paving the way for genetic counseling to help affected families make informed medical and personal decisions.

Life-long estrogen exposure is one of the major risk factors in the development and progression of breast cancer. However, little is known about the molecular mechanisms, by which chronic exposure to estrogen contributes to breast carcinogenesis. The aim of the present study was to investigate the effects of long-term exposure with 4-hydroxyestradiol (4-OHE2) on acquired cancer characteristics of human mammary epithelial MCF-10A cells. The possible regulators were further studied in chronic 4-OHE2-treated MCF-10A cells. We observed that MCF-10A cells long-term exposed to 4-OHE2 acquire the characteristics of cancer cells, such as enhanced cell growth, EMT properties, and increased migration and invasiveness. Moreover, the expression of CYP1B1 was significantly elevated in long-term 4-OHE2-treated MCF-10A cells. Block of CYP1B1 significantly reduced the cancer cell characteristics in long-term 4-OHE2-treated MCF-10A cells. Our results indicated that 4-OHE2 mediated enhanced cancer cell characteristics in mammary epithelial cells are an important key event for breast carcinogenesis process. CYP1B1 partially contributes to the 4-OHE2 induced cancer cell characteristics in MCF-10A cells. Targeting CYP1B1 might offer a new strategy for the treatment of estrogen-induced breast cancer.

Aim The aryl hydrocarbon receptor (AhR)-ligand axis has been shown to be involved in inflammatory diseases and bone homeostasis. However, the activation of AhR signalling pathway and the possible functions of AhR ligands in periodontitis are underexplored. This study investigated the expression of the AhR target gene cytochrome P450 subfamily B member 1 (CYP1B1) and the functions and mechanisms of the AhR ligand 6 formylindolo[3,2-b]carbazole (FICZ) in periodontitis. Materials and methods CYP1B1 expression was detected in human periodontitis samples, mice with ligature-induced periodontitis and lipopolysaccharide (LPS)-induced inflammation in periodontal ligament cells (PDLCs) in vitro. FICZ was administered topically or systemically. The therapeutic functions of FICZ were detected via qPCR, micro-computed tomography and immunohistochemistry. Finally, the mechanisms of AhR signalling in periodontitis were investigated by cell assays. Results CYP1B1 expression was downregulated in periodontitis. FICZ rescued the alveolar bone loss and mitigated the inflammatory cytokines in periodontitis mice. In vitro, FICZ pre-treatment reduced the LPS-induced inflammation in PDLCs via the increased phosphorylation of STAT3. Additionally, FICZ prompted the mineralization of PDLCs via activation of the Wnt/β-catenin signalling pathway. Conclusion AhR signalling pathway is suppressed in periodontitis and the AhR ligand FICZ can prevent periodontitis.

Breast cancer in men is a rare and still poorly characterized disease. Inherited mutations in BRCA1, BRCA2 and PALB2 genes, as well as common polymorphisms, play a role in male breast cancer genetic predisposition. Male breast cancer is considered a hormone-dependent tumor specifically related to hyperestrogenism. Polymorphisms in genes involved in estrogen biosynthesis and metabolism pathways, such as CYP17A1 and CYP1B1, have been associated with breast cancer risk. Here, we aimed to investigate the role of CYP17A1 and CYP1B1 polymorphisms in male breast cancer risk. A series of 597 male breast cancer cases and 1022 male controls, recruited within the Italian Multicenter Study on male breast cancer, was genotyped for CYP17A1 rs743572, CYP1B1 rs1056836 and rs1800440 polymorphisms by allelic discrimination real-time PCR with TaqMan probes. Associations with male breast cancer risk were estimated using logistic regression. No statistically significant associations between male breast cancer risk and the three analyzed polymorphisms emerged. Similar results were obtained also when BRCA1/2 mutational status was considered. No significant differences in the distribution of the genotypes according to estrogen receptor status emerged. In conclusion, our study, based on a large series of male breast cancer cases, is likely to exclude a relevant role of CYP17A1 and CYP1B1 polymorphisms in male breast cancer predisposition. Overall, these results add new data to the increasing evidence that polymorphisms in these genes may not be associated with breast cancer risk.

Based on a small number of cases, interferon beta (IFN-β) has been added to the list of drugs that might induce pulmonary arterial hypertension (PAH) in the current European guidelines for the diagnosis and treatment of pulmonary hypertension. Here, we propose that multiple sclerosis patients who are genetically predisposed to PAH may be at higher risk to develop disease when treated with IFN-β. We included two patients with multiple sclerosis who developed a manifest PAH after five amd eight years on IFN-β 1a therapy, respectively (without confirmed right heart catheterization). In both patients, PAH markedly improved after discontinuation of IFN-β 1a and initiation of targeted PAH therapy. For genetic analysis, we used a PAH-gene panel based on next-generation sequencing of 16 PAH and 38 candidate genes. In one of the two patients, we could identify a nonsense variant in the PAH gene ATP13A3 . The second patient showed a missense variant of the CYP1B1 gene, which might be linked to PAH predisposition. The results of this study support the hypothesis that multiple sclerosis patients who receive IFN-β 1a therapy might be at higher risk for the development of manifest PAH, if they carry a pathogenic variant or sequence variant genetically predisposing to the disease. However, further studies are necessary to systematically investigate the presence of predisposing PAH gene variants in these patients.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. As part of a programme to develop anticancer prodrugs which are activated by cytochrome P450 (CYP)1B1, a library of 4,6-diaryl-2-pyridones was synthesised in yields of 6-60% from the corresponding chalcones. A number of these derivatives showed promising antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1, while showing little toxicity towards a non-tumour breast cell line with no CYP expression. Metabolism studies provided evidence supporting the involvement of CYP1 enzymes in the bioactivation of these compounds.

AIM: To identify the novel mutation alleles in the CYP1B1 gene of primary congenital glaucoma (PCG) patients at Shandong Province of China, and investigate their correlation with glaucomatous features. METHODS: The DNA from the peripheral blood of 13 congenital glaucoma patients and 50 ethnically matched healthy controls from the affiliated hospital of Qingdao University were extracted. The coding region of the CYP1B1 gene was amplified by PCR and direct DNA sequencing was performed. Disease causing-variants were analyzed by comparing the sequences and the structures of wild type and mutant CYP1B1 proteins by PyMOL software. RESULTS: Two missense mutations, including A330F caused by c.988G>T&c.989C>T, and R390H caused by c.1169G>A, were identified in one of the 13 PCG patients analyzed in our study. A330F mutation was observed to be novel in the Chinese Han population, which dramatically altered the protein structure of CYP1B1 gene, including the changes in the ligand-binding pocket. Furthermore, R390H mutation caused the changes in heme-protein binding site of this gene. In addition, the clinical phenotype displayed by PCG patient with these mutations was more pronounced than other PCG patients without these mutations. Multiple surgeries and combined drug treatment were not effective in reducing the elevated intraocular pressure in this patient. CONCLUSION: A novel A330F mutation is identified in the CYP1B1 gene of Chinese PCG patient. Moreover, in combination with other mutation R390H, this PCG patient shows significant difference in the CYP1B1 protein structure, which may specifically contribute to severe glaucomatous phenotype.

Breast cancer is the most common malignancy among women worldwide. Various studies indicate that prolonged exposure to elevated levels of estrogens is associated with development of breast cancer. Both estrogen receptor-dependent and independent mechanisms can contribute to the carcinogenic effects of estrogens. Among them, the oxidative metabolism of estrogens plays a key role in the initiation of estradiol-induced breast cancer by generation of reactive estrogen quinones as well as the associated formation of oxygen free radicals. These genotoxic metabolites can react with DNA to form unstable DNA adducts which generate mutations leading to the initiation of breast cancer. A variety of endogenous and exogenous factors can alter estrogen homeostasis and generate genotoxic metabolites. The use of specific phytochemicals and dietary supplements can inhibit the risk of breast cancer not only by the modulation of several estrogen-activating enzymes (CYP19, CYP1B1) but also through the induction of various cytoprotective enzymes (eg, SOD3, NQO1, glutathione S-transferases, OGG-1, catechol-O-methyltransferases, CYP1B1A, etc.) that reestablish the homeostatic balance of estrogen metabolism via nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent and independent mechanisms.

Cytochrome P450 (CYPs) 1 family is an important phase Ⅰ enzyme involved in carcinogen activation. Nitidine chloride (NC) is a pharmacologically active alkaloid with polyaromatic hydrocarbon found in the roots of Zanthoxylum nitidum (Roxb.) DC, a traditional medicinal herb widely used in China. We examined the inhibitory effects of NC on CYPs1A1, 1B1, and 1A2. NC significantly inhibited CYPs1A1 and 1B1-catalyzed ethoxyresorufin O-deethylation (EROD) activity (IC50 = 0.28 ± 0.06 and 0.32 ± 0.02 μM, respectively) in a concentration-dependent manner, but only showed slight inhibition of CYP1A2 activity (IC50 ＞ 50 μM). Kinetic analysis revealed that NC competitively inhibited CYP1B1 with Ki of 0.47 ± 0.05 μM, whereas NC caused a mixed type of inhibition on CYP1A1 with Ki and KI of 0.14 ± 0.04 and 0.19 ± 0.09 μM, respectively. The observed enzyme inhibition neither required NADPH nor revealed time dependency. Molecular docking manifested the generation of strong hydrogen-bonding interactions of Ser116 of CYP1A1 and Ser127 of CYP1B1 with methoxy moiety of NC. Additionally, NC-induced alteration of estradiol (E2) metabolism was also investigated in the present study. Hydroxyestradiols, including 2-hydroxyestradiol (nontoxic) and 4-hydroxyestradiol (genotoxic) generated in recombinant enzyme incubation systems and cultured MCF-7 cells were analyzed, and NC was found to preferentially inhibit the nontoxic 2-hydroxylation activity of E2 mediated by CYP1A1. In conclusion, NC was a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. The remarkable inhibition on E2 2-hydroxylation might increase the risk of 4-OHE2-induced genotoxicity. SIGNIFICANCE STATEMENT CYP1 enzymes catalyze oxidative metabolism of a variety of compounds and are known to play a crucial role in the development of cancer. CYP1A1 and CYP1A2 are responsible for hydroxylation of estradiol (E2) at C-2 position, resulting in the formation of 2-hydroxyestradiol (2-OHE2), which is proposed to be a detoxification pathway. However, CYP1B1-mediated hydroxylation of E2 at C-4 position has been suggested to be a tumor initiator. The present study found that nitidine chloride (NC) is a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. NC preferentially inhibited the nontoxic E2 2-hydroxylation pathway mediated by CYP1A1, which might increase the risk of 4-OHE2-induced genotoxicity and cause severe drug-drug interactions.

Polycyclic aromatic hydrocarbons (PAHs) comprise a large family of toxic compounds that come from natural and anthropogenic sources. Chrysene is a PAH with multiple effects, but the toxic potentials of mono-methylchrysenes are less characterized. A comparison of chrysene and six mono-methylchrysenes was performed using assays for cytotoxicity, human aryl hydrocarbon receptor (AhR) reporter gene signaling, and AhR-regulated target gene and protein expression. Sulforhodamine B and trypan blue dye binding assays revealed these chrysenes to be similar in their cytotoxic effects on HepG2 cells. A yeast-based reporter assay detecting human AhR-mediated gene expression identified 4-methylchrysene as being six times more potent and 5-methylchrysene about one-third as potent as chrysene. Other methylchrysenes were more similar to chrysene in the ability to act as AhR ligands. The mono-methylchrysenes all strongly induced CYP1A1 mRNA and protein and moderately induced CYP1B1 expression in HepG2 cells. Levels of CYP1A2 mRNA were induced at higher concentrations of the chrysenes, but protein expression was not significantly altered. The PCR-based gene expression and immunoblotting analyses indicated induced expression differences across the chrysene members were similar to each other. Overall, the effects of methylated chrysenes were comparable to unsubstituted chrysene, suggesting members of this group may be considered approximately equivalent in their effects. © 2019 Wiley Periodicals, Inc.

Environmental pollutants including halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons, including benzo[a]pyrene, exert their deleterious effects through the activation of the aryl hydrocarbon receptor (AHR) and by the resulting transcription of genes not yet fully identified. Ligand-bound AHR translocates from cytoplasm to nucleus, where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. The AHR/ARNT dimer binds to enhancer regions of responsive genes to activate transcription. AHR also mediates carcinogenesis caused by PAHs, likely via CYP1A1, CYP1A2, and CYP1B1, which are massively induced by activated AHR in many tissues and generate carcinogenic electrophilic derivatives of PAHs. In the current study, we have used the mouse GeCKOv2 genome-wide CRISPR/Cas9 library to identify novel genes in the AHR pathway by taking advantage of a B[a]P selection assay that we previously used to identify core AHR pathway genes in Hepa-1c1c7 murine hepatoma cells. Besides Ahr, Arnt, and Cyp1a1, we report the identification of multiple additional putative AHR pathway genes including several that we validated. These include cytochrome P450 reductase (Por), which mediates redox regeneration of cytochromes P450, and 5 genes of the heme biosynthesis pathway: delta-aminolevulinate synthase 1 (Alas1), porphobilinogen deaminase (Hmbs), uroporphyrinogen decarboxylase (Urod), coproporphyrinogen oxidase (Cpox), and ferrochelatase (Fech): heme being an essential prosthetic group of cytochrome P450 proteins. Notably, several of these genes were identified by GeCKO screening, despite not being identifiable by reverse genetics approaches. This indicates the power of high-sensitivity genome-wide genetic screening for identifying genes in the AHR pathway.

Background/aim Benzo[a]pyrene (BaP), an environmental pollutant produced by combustion processes, induces expression of cytochrome P450 (CYP) 1A1 via the activation of aryl hydrocarbon receptor (AHR). Induced CYP1A1 is involved in BaP metabolism, resulting in either detoxification or metabolic activation in a context-dependent manner. The effect of diallyl trisulfide (DATS), a garlic-derived organosulfur compound, on BaP metabolism has not been investigated. Materials and methods The combined effect of DATS and BaP on BaP metabolism in hepatocyte-derived HepG2 cells was examined. Results DATS enhanced BaP-induced CYP1A1 and CYP1B1 mRNA expression, BaP hydroxylation and BaP-DNA adduct formation. Combined treatment of BaP and DATS also increased reactive oxygen species levels. DATS enhanced BaP-induced AHR recruitment and histone H3 acetylation on the CYP1A1 promoter. Conclusion DATS combined treatment enhances BaP metabolic activation through an AHR-modulating mechanism.

Aryl Hydrocarbon Receptor (AhR) is a key player to regulate the expression of a group of enzymes known as cytochrome P450s (CYPs) super family (CYP1A1, CYP1B1, CYP2B6, and CYP2E1) which metabolites...

Ethnopharmacological relevance Zuotai (gTso thal) has a long history in the treatment of cardiovascular disease, liver and bile diseases, spleen and stomach diseases as a precious adjuvant in Tibetan medicine. However, Zuotai is a mercury preparation that contains 54.5% HgS. Its application has always been controversial. Aim of the study To evaluate the toxicological effects of Zuotai in hepatocytes and in zebrafish. Materials and methods MTT was used to determine the survival rate of hepatocytes; Hoechst and TUNEL staining were used to detect the apoptosis cells; Western blot and RT-qPCR assay were used to determine the expression levels of the protein and mRNA; Liver morphology observation and H&E staining were used to evaluate the hepatotoxicity of Zuotai in Zebfrafish. Results The survival rate of L-02 cells, HepG2 cells and RBL-2A cells reduced by Zuotai (10−4–0.1 mg/mL) in a dose and time-dependent manner. Zuotai (0.1 mg/mL) induced HepG2 cells shrinkage, condensation and fragmentation and increased the number of apoptosis cells. The protein expression levels of cleaved Caspase-3 and Bax were increased and the expression levels of Bcl-2 were reduced after HepG2 cells exposed to Zuotai (10−4–0.1 mg/mL) for 24 h. In addition, Zuotai (0.2 mg/mL) induced the darker liver color of the larval zebrafish and changed the liver morphologic of adult zebrafish. Zuotai (0.2 mg/mL) also increased the mRNA levels of CYP1A1, CYP1B1 and MT-1 in the liver of adult zebrafish. However, no significantly hepatotoxicity was observed after hepatocytes and zebrafish exposed to HgS at the same dose. Conclusions Results showed that Zuotai induced hepatotoxicity effectively under a certain dose but its hepatotoxicity likely occurs via other mechanisms that did not depend on HgS.

Recent studies suggest that HIV infection is an independent risk factor for the development of chronic obstructive pulmonary disease (COPD). We hypothesized that HIV infection and cigarette smoking synergize to alter the function of alveolar macrophages (AMs). To test this hypothesis, global transcriptome analysis was performed on purified AMs from 20 individuals split evenly between HIV-uninfected nonsmokers and smokers and untreated HIV-infected nonsmokers and smokers. Differential expression analysis identified 143 genes significantly altered by the combination of HIV infection and smoking. Of the differentially expressed genes, chitinase 1 (CHIT1) and cytochrome P450 family 1 subfamily B member 1 (CYP1B1), both previously associated with COPD, were among the most upregulated genes (5- and 26-fold, respectively) in the untreated HIV-infected smoker cohort compared with HIV-uninfected nonsmokers. Expression of CHIT1 and CYP1B1 correlated with the expression of genes involved in extracellular matrix organization, oxidative stress, immune response, and cell death. Using time-of-flight mass cytometry to characterize AMs, a significantly decreased expression of CD163, an M2 marker, was seen in HIV-infected subjects, and CD163 inversely correlated with CYP1B1 expression in AMs. CHIT1 protein levels were significantly upregulated in bronchoalveolar lavage fluid from HIV-infected smokers, and increased CHIT1 levels negatively correlated with lung function measurements. Overall, these findings raise the possibility that elevated CHIT1 and CYP1B1 are early indicators of COPD development in HIV-infected smokers that may serve as biomarkers for determining this risk.

Graphical abstract, Highlights • AHR is over-expressed and hyperactivated in carcinoma tissues of IBC patients. • AHR knockdown inhibits expression of CYP1B1 and Wnt5a in IBC cells. • AHR and CYP1B1 expression correlates with Wnt5 a/b and b-catenin expression levels. • AHR and CYP1B1 expression correlates with percentage of CD44(+)/CD24(−/low) subset in IBC. • AHR and its surrogate molecules correlate with IBC poor prognosis., The aim of the present study was to evaluate the expression levels of the aryl hydrocarbon receptor (AHR) and its target gene CYP1B1 and to correlate their expression with Wnt5a/b-β-catenin, the CD44+/CD24(−/low) cancer stem cell (CSC) subset and factors associated with poor prognosis in inflammatory breast cancer (IBC) and non-IBC patients. The methods of analysis used were quantitative real-time PCR, western blotting, immunohistochemistry and flow cytometry. Compared to non-IBC tissues, IBC tissues exhibited the overexpression of AHR and its target gene/protein CYP1B1. AHR and CYP1B1 mRNA levels were associated with the poor clinical prognosis markers tumour grade, lymphovascular invasion, cell proliferation and lymph node metastasis. Furthermore, AHR expression correlated with the expression of Wnt5a/b and β-catenin signalling molecules, and Wnt5a mRNA expression was downregulated in the SUM149 human IBC cell line and the MDA-MB-231 non-IBC cell line upon inhibition of AHR. AHR gene knockout (CRISPR-Cas9) inhibits CYP1B1 and Wnt5a expression in the IBC cell line. The CD44+/CD24(−/low) subset was significantly correlated with the expression of AHR, CYP1B1, Wnt5a/b and β-catenin in IBC tissues. The overexpression of AHR and its target CYP1B1 correlated with the expression of Wnt5a/b and β-catenin, CSCs, and poor clinical prognostic factors of IBC. Thus, targeting AHR and/or its downstream target molecules CYP1B1 and Wnt5a/b may represent a therapeutic approach for IBC.

The most common cancer in women is breast cancer, and it poses serious health issues in women, leading to increased mortality rates. Estrogen and its metabolites have been established as risk factors for breast cancer. Determining the role of genetic variants having functional involvement in regulating the estrogen concentration and detoxifying the toxic estrogen metabolites through estrogen hydroxylation by CYP1A1, CYP1B1, and catechol estrogen inactivation by SULT1A1 may reveal crucial information on the identification of genetic biomarkers for breast cancer susceptibility, thus paving the way for monitoring, early detection, and personalized treatment. In the present study, 200 breast cancer and 200 control samples were analyzed with regard to genotypes for the polymorphism rs4646903, rs1048943, rs1056836, and rs9282861 found in the genes CYP1A1, CYP1B1, and SULT1A1, respectively, by real-time polymerase chain reaction. The study revealed positive association of CYP1A1 rs4646903 T to C polymorphism with breast cancer risk. A significant increase of TC and CC alleles of the gene CYP1A1 m1(T/C) genotype rs4646903 was found in patients in comparison with controls (OR = 1.68; 95% CI, 1.13–2.51; p: 0.009). A significant increase of GA and AA alleles of the SULT1A1 rs9282861 genotype was found in patients in comparison with controls (OR = 1.86; 95% CI, 1.21–2.86; p: 0.004). SULT1A1 Arg213His was found to contribute an elevated risk of breast cancer. There was no strong and significant linkage disequilibrium between these polymorphisms. CYP1A1 and SULT1A1 were found to be associated with premenopausal breast cancer cases and high tumor grade. Stratified analysis of the subjects based on receptor status revealed an association between SULT1A1 polymorphism and ER-positive breast cancer cases. The in silico analysis revealed structural variation in SULT1A1 gene with respect to rs9282861 polymorphism. The polymorphisms rs9282861 of SULT1A1 and rs4646903 of CYP1A1 were found to be associated with breast cancer risk in southern Indian population.

Human cytochrome P450 1B1 (CYP1B1)-mediated formation of 4-hydroxyestradiol (4-OHE2) from 17β-estradiol plays an important role in the progression of human breast cancer, while the biotransformation of 17β-estradiol to 2-hydroxyestradiol mediated by cytochrome P450 1A1 (CYP1A1) is considered as a less harmful pathway. In this study, inhibitory effects of flavonoids baicalein and oroxylin A, a metabolite of baicalein in human body, on CYP1A1 and 1B1 activities were investigated in vitro. The inhibition intensities of baicalein and oroxylin A towards CYP1B1 were greater than towards CYP1A1 with a mixed mechanism. In addition, oroxylin A showed a stronger inhibitory effect than baicalein towards the CYP1B1-mediated 17β-estradiol 4-hydroxylation, with the IC50 values of 0.0146 and 2.27 μM, respectively. Docking studies elucidated that oroxylin A had a stronger binding affinity than baicalein for CYP1B1. In MCF-7 cells, compared with baicalein-treated groups, oroxylin A with lower doses decreased and increased the formation of 4-OHE2 and 2-hydroxyestradiol, respectively, with a preferential induction of mRNA of CYP1A1 over CYP1B1. In conclusion, this study demonstrated that oroxylin A showed a stronger inhibitory effect than baicalein on CYP1B1-mediated 4-OHE2 formation in MCF-7 cells, providing crucial implications for their possibly preventive/therapeutic potential against breast cancer via inhibition of CYP1B1, particularly of oroxylin A.

Background: In addition to its role as a broad-spectrum imidazole antifungal drug, data from animal models as well as human clinical trials also demonstrated an anti-inflammatory efficacy of bifonazole (BFZ). In the histamine wheal test and after UV radiation, BFZ showed antiphlogistic effects that were comparable to those of hydrocortisone. However, the underlying molecular mechanisms of the anti-inflam­matory properties of BFZ are poorly understood. Methods: Performing an in vitro study we used full-thickness three-dimensional (3D) skin models containing macrophages as mediators of inflammation. We conducted two sets of experiments. In a first set we exposed our models to UVB irradiation to provoke an inflammation. A second approach used the addition of histamine into the culture medium. In both approaches, models were treated topically with a BFZ-containing ointment or a placebo ointment for 24 h, and then the effects were examined histologically as well as with microarray and quantitative real-time PCR analyses. Results: Histological examination showed that the BFZ-containing ointment reconstituted UVB- and histamine-mediated disorders within the skin models. Performing gene expression profiling in models that were treated with the BFZ-containing ointment after UVB irradiation, we detected an upregu­lation of differentiation markers (fillagrin, loricrin, and keratin 1), antimicrobial peptides (DEFB103A), and members of the cytochrome P450 family (CYP1A1 and CYP1B1) as well as a downregulation of genes that are involved in immune response (CCL22, CXCL12, CCL7, IRF1, ICAM1, TLR3, and RARRES3) and matrix metalloproteinases (MMP12 and MMP7). Models that were treated with the BFZ-containing ointment after histamine application showed an upregulation of members of the cytochrome P450 family (CAP1A1, CYP1B1, and CYP24A1) and a downregulation of immune response-associated genes (CXCL6, CXCL12, CCL8, IL6, and IL32). Conclusion: We present the first in vitro study showing anti-inflammatory effects of BFZ in human 3D skin models. To our knowledge, this is the first time that these effects could be translated from human clinical trials into an in vitro test system, allowing a more detailed examination of molecular mechanisms that were regulated by BFZ.

Increasing use of zebrafish in biomedical, toxicological and developmental studies requires explicit knowledge of cytochrome P450 (CYP), given the central role of CYP in oxidative biotransformation of xenobiotics and many regulatory molecules. A full complement of CYP genes in zebrafish and their transcript expression during early development have already been examined. Here we established a comprehensive picture of CYP gene expression in the adult zebrafish liver using a RNA-seq technique. Transcriptional profiling of a full complement of CYP genes revealed that CYP2AD2, CYP3A65, CYP1A, CYP2P9 and CYP2Y3 are major CYP genes expressed in the adult zebrafish liver in both sexes. Quantitative real-time RT-PCR analysis for selected CYP genes further supported our RNA-seq data. There were significant sex differences in the transcript levels for CYP1A, CYP1B1, CYP1D1 and CYP2N13, with males having higher expression levels than those in females in all cases. A similar feature of gender-specific expression was observed for CYP2AD2 and CYP2P9, suggesting sex-specific regulation of constitutive expression of some CYP genes in the adult zebrafish liver. The present study revealed several "orphan" CYP genes as dominant isozymes at transcript levels in the adult zebrafish liver, implying crucial roles of these CYP genes in liver physiology and drug metabolism. The current results establish a foundation for studies with zebrafish in drug discovery and toxicology.

The expression of potential prognostic markers of human colon adenocarcinoma in tumor and adjacent normal tissue, as well as the relationship between their expression levels was studied. The expression of potential prognostic markers (IGFBP3, AhR, CYP1A1, CYP1B1, HIF-1α, OCT4A, OCT4B, OCT4B1) was assayed with quantitative RT-PCR (qPCR). A correlation between IGFBP3 and AhR expression was found. AhR signaling was analyzed in the primary culture of human colon adenocarcinoma BSK 8 with IGFBP3 gene knockout. It was found that the enhanced expression of AhR can be an additional diagnostic criterion for colon cancer. The correlation between the expression level of HIF-1α, IGFPB3, and OCT4 isoforms and clinic stage (TNM) was not estimated. Clones with IGFBP3 gene knockout had augmented doubling time and increased resistance to the cytostatic drug 5-ftoruracile after modulated activity of dioxin receptor with its agonist—indol-3-carbinol.

Humans are exposed to phthalates daily via items such as personal care products and medications. Reproductive toxicity has been documented in mice exposed to di-n-butyl phthalate (DBP); however, quantitative evidence of its metabolite, mono-n-butyl phthalate (MBP), reaching the mouse ovary and its effects on hepatic and ovarian biotransformation enzymes in treated mice is still lacking. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was employed to quantify MBP levels in liver, serum, and ovary from mice treated with a single or repeated exposure to the parent compound, DBP. Adult CD-1 females were pipet fed once or for 10 days with vehicle (tocopherol-stripped corn oil) or DBP at 1, 10, and 1000 mg/kg/day. Tissues and serum were collected at 2, 6, 12, and 24 h after the single or final dose and subjected to LC-MS/MS. Ovaries and livers were processed for qPCR analysis of selected phthalate-associated biotransformation enzymes. Regardless of duration of exposure (single vs repeated), MBP was detected in the tissues of DBP-treated mice. In single dose mice, MBP levels peaked at ≤6 h and fell close to background levels by 24 h post-exposure. Following the last repeated dose, MBP levels peaked at ≤2 h and fell to background levels by 12 h. Hepatic and ovarian expression of Lpl, Aldh1a1, Adh1, Ugt1a6a, and Cyp1b1 were altered in DBP-treated mice in a time- and dose-specific manner. These findings confirm that MBP reaches the mouse liver and ovary after oral exposure to DBP and influences the expression of hepatic and ovarian phthalate-associated biotransformation enzymes.

Background At present, the role of lncRNAs in the pathogenesis of hypoxia-induced pulmonary hypertension (HPH) is not fully understood. This study aimed to explore differences in the hypoxia-induced expression of lncRNAs and their potential role in multiple pulmonary artery cells. Methods LncRNA expression in pulmonary artery smooth muscle cells (PASMCs), pulmonary microvascular endothelial cells (PMECs), and pericytes (PCs) was analyzed by high-throughput sequencing and compared between normoxic and hypoxic cells. Bioinformatics analysis was conducted to predict their functions. Results PASMCs, PMECs, and PCs displayed 275 (140 upregulated), 251 (162 upregulated), and 290 (176 upregulated) different lncRNAs, respectively. Among these, lncRNA TUG1 levels increased in PASMCs and PCs but decreased in PMECs. Bioinformatics analysis indicated that lncRNA TUG1 might target miR-145-5p, thereby affecting SOX4 and BMF expression, and could also regulate miR-129-5p levels to affect CYP1B1 and VCP expression. It could also regulate miR-138-5p levels to affect KCNK3 and RHOC expression. Conclusions Hypoxia exposure of vascular cells resulted in differential expression of lncRNAs, especially lncRNA TUG1, which showed significant abnormal expression in all three types of vascular cells under hypoxia. Our results suggested that abnormal expression of lncRNA TUG1 might be involved in the regulation of pulmonary vascular cell function under hypoxia.

Background and Aims: The genes involved in detoxification and the elimination of toxic metabolites have a vital role in cancer pathogenesis. Also, there is evidence that higher amounts of body fat are associated with increased risks of several cancers. The current study aims to identify the relationship of age, body mass index (BMI), smoking, family history, and polymorphism rs1056827 of CYP1B1 with prostate cancer. Materials and Methods: A total of 103 patients and 103 healthy men as control groups were enrolled in the current study. The DNAs were extracted using the salting-out method after venesection, and the genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. The obtained data were analyzed by SPSS version 23 using X2 statistical tests and logistic regression. Results: The results showed a significant association between the study groups regarding age, BMI, and family history of the disease (p = 0.024, p = 4.08⨯10-4, and p = 3.58⨯10-19, respectively). Investigation of genetic models except to additive model showed a significant relationship between TT (Ser/Ser) genotype and prostate cancer. It also showed a strong association between this genotype with BMI and family history. Conclusion: The current study results showed that the carriers of the TT genotype with a high BMI have a higher chance of developing prostate cancer. Further studies in this area will provide stronger results.

Breast cancer (BC) is a leading cause of cancer deaths in women in less developed countries and the second leading cause of cancer death in women in the U.S. In this study, we report the inhibition of E2-mediated mammary tumorigenesis by Cuminum cyminum (cumin) administered via the diet as cumin powder, as well as dried ethanolic extract. Groups of female ACI rats were given either an AIN-93M diet or a diet supplemented with cumin powder (5% and 7.5%, w/w) or dried ethanolic cumin extract (1%, w/w), and then challenged with subcutaneous E2 silastic implants (1.2 cm, 9 mg). The first appearance of a palpable mammary tumor was significantly delayed by both the cumin powder and extract. At the end of the study, the tumor incidence was 96% in the control group, whereas only 55% and 45% animals had palpable tumors in the cumin powder and extract groups, respectively. Significant reductions in tumor volume (660 ± 122 vs. 138 ± 49 and 75 ± 46 mm3) and tumor multiplicity (4.21 ± 0.43 vs. 1.16 ± 0.26 and 0.9 ± 0.29 tumors/animal) were also observed by the cumin powder and cumin extract groups, respectively. The cumin powder diet intervention dose- and time-dependently offset E2-related pituitary growth, and reduced the levels of circulating prolactin and the levels of PCNA in the mammary tissues. Mechanistically, the cumin powder diet resulted in a significant reversal of E2-associated modulation in ERα, CYP1A1 and CYP1B1. Further, the cumin powder diet reversed the expression levels of miRNAs (miR-182, miR-375, miR-127 and miR-206) that were highly modulated by E2 treatment. We analyzed the composition of the extract by GC/MS and established cymene and cuminaldehyde as major components, and further detected no signs of gross or systemic toxicity. Thus, cumin bioactives can significantly delay and prevent E2-mediated mammary tumorigenesis in a safe and effective manner, and warrant continued efforts to develop these clinically translatable spice bioactives as chemopreventives and therapeutics against BC.

Metformin (MET) is a clinically used anti-hyperglycemic agent that shows activities against chemically-induced animal models of cancer. A study from our laboratory showed that MET protectes against 7, 12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis in vitro human non-cancerous epithelial breast cells (MCF10A) via activation of the aryl hydrocarbon receptor (AhR). However, it is unclear whether MET can prevent the initiation of breast carcinogenesis in an in vivo rat model of AhR-induced breast carcinogenesis. Therefore, the main aims of this study are to examine the effect of MET on protecting against rat breast carcinogenesis induced by DMBA and to explore whether this effect is medicated through the AhR pathway. In this study, treatment of female rats with DMBA initiated breast carcinogenesis though inhibiting apoptosis and tumor suppressor genes while inducing oxidative DNA damage and cell cycle proliferative markers. This effect was associated with activation of AhR and its downstream target genes; cytochrome P4501A1 (CYP1A1) and CYP1B1. Importantly, MET treatment protected against DMBA-induced breast carcinogenesis by restoring DMBA effects on apoptosis, tumor suppressor genes, DNA damage, and cell proliferation. Mechanistically using in vitro human breast cancer MCF-7 cells, MET inhibited breast cancer stem cells spheroids formation and development by DMBA, which was accompanied by a proportional inhibition in CYP1A1 gene expression. In conclusion, the study reports evidence that MET is an effective chemopreventive therapy for breast cancer by inhibiting the activation of CYP1A1/CYP1B1 pathway in vivo rat model.

Downregulation of drug metabolizing enzymes and transporters by proinflammatory mediators in hepatocytes, enterocytes and renal tubular epithelium is an established mechanism affecting pharmacokinetics. Emerging evidences indicate that vascular endothelial cell expression of drug metabolizing enzymes and transporters may regulate pharmacokinetic pathways in heart to modulate local drug bioavailability and toxicity. However, whether inflammation regulates pharmacokinetic pathways in human cardiac vascular endothelial cells remains largely unknown. The lipid modified protein Wnt5A is emerging as a critical mediator of proinflammatory responses and disease severity in sepsis, hypertension and COVID-19. In the present study, we employed transcriptome profiling and gene ontology analyses to investigate the regulation of expression of drug metabolizing enzymes and transporters by Wnt5A in human coronary artery endothelial cells. Our study shows for the first time that Wnt5A induces the gene expression of CYP1A1 and CYP1B1 enzymes involved in phase I metabolism of a broad spectrum of drugs including chloroquine (the controversial drug for COVID-19) that is known to cause toxicity in myocardium. Further, the upregulation of CYP1A1 and CYP1B1 expression is preserved even during inflammatory crosstalk between Wnt5A and the prototypic proinflammatory IL-1β in human coronary artery endothelial cells. These findings stimulate further studies to test the critical roles of vascular endothelial cell CYP1A1 and CYP1B1, and the potential of vascular-targeted therapy with CYP1A1/CYP1B1 inhibitors in modulating myocardial pharmacokinetics in Wnt5A-associated inflammatory and cardiovascular diseases.

The aryl hydrocarbon receptor (AhR) acts as a receptor that responds to ligands, including dioxin. The AhR–ligand complex translocates from the cytoplasm into the nucleus to induce gene expression. Because dioxin exposure impairs cognitive and neurobehavioral functions, AhR-expressing neurons need to be identified for elucidation of the dioxin neurotoxicity mechanism. Immunohistochemistry was performed to detect AhR-expressing neurons in the mouse brain and confirm the specificity of the anti-AhR antibody using Ahr−/− mice. Intracellular distribution of AhR and expression level of AhR-target genes, Cyp1a1, Cyp1b1, and Ahr repressor (Ahrr), were analyzed by immunohistochemistry and quantitative RT-PCR, respectively, using mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The mouse brains were shown to harbor AhR in neurons of the locus coeruleus (LC) and island of Calleja major (ICjM) during developmental period in Ahr+/+ mice but not in Ahr−/− mice. A significant increase in nuclear AhR of ICjM neurons but not LC neurons was found in 14-day-old mice compared to 5- and 7-day-old mice. AhR was significantly translocated into the nucleus in LC and ICjM neurons of TCDD-exposed adult mice. Additionally, the expression levels of Cyp1a1, Cyp1b1, and Ahrr genes in the brain, a surrogate of TCDD in the tissue, were significantly increased by dioxin exposure, suggesting that dioxin-activated AhR induces gene expression in LC and ICjM neurons. This histochemical study shows the ligand-induced nuclear translocation of AhR at the single-neuron level in vivo. Thus, the neurotoxicological significance of the dioxin-activated AhR in the LC and ICjM warrants further studies.

Xiaochaihu decoction is one of the most important traditional Chinese medicines that is widely used with other drugs in clinical practice, and may cause drug-drug interactions. However, there is not sufficient experimental evidence for the effects of Xiaochaihu decoction on cytochrome P450s (CYPs). The aim of the present study was to investigate the effects of Xiaochaihu decoction on the mRNA and protein levels of hepatic CYPs. Eighty normal male Sprague-Dawley (SD) rats were randomly divided into two groups based on body weight and duration of drug administration (3 and 6 days). Each group was further divided into subgroups: Control group (2 ml 5‰ CMC-Na); hepatic enzyme inducer group (50 mg/kg/day rifampicin); and experimental groups (Xiaochaihu decoction: Low dose, 1.7 g/kg/day; medium dose, 3.4 g/kg/day; high dose, 6.8 g/kg/day). The effects of Xiaochaihu decoction on Cyp1a2, Cyp3a1, Cyp2d6, and Cyp1b1 mRNA and protein expression in rats were evaluated using reverse transcription quantitative reverse transcription polymerase chain reaction and western blot analysis. After 3 days, medium dose of Xiaochaihu decoction inhibited the mRNA and protein expression of Cyp1a2, Cyp3a1 and Cyp1b1. In addition, after 6 days, Xiaochaihu decoction induced Cyp3a1 mRNA expression at low and medium doses; Cyp2d6 mRNA expression at low and high doses; and Cyp2d6 protein expression at high doses. Nonetheless, the gene and protein expression of Cyp1b1 was not affected at any dose. The findings of the present study may provide insights into potential drug-drug interactions associated with Xiaochaihu decoction.