CYP1B1 mRNA Levels Research Articles

Taxifolin alleviates apoptotic injury induced by DEHP exposure through cytochrome P450 homeostasis in chicken cardiomyocytes

Di-2-ethylhexyl phthalate (DEHP), widely used as a plasticizer, is a ubiquitous artificial pollutant. DEHP can induce biological toxicity in various organs, with an especially high potential for toxicity to the cardiovascular system. Taxifolin (TAX) is used in the treatment of cardiovascular diseases due to its antioxidative capacities. However, it is not clear whether TAX can alleviate apoptosis induced by DEHP exposure through the cytochrome P450 (CYP) pathway in cardiomyocytes. To understand the role of TAX in attenuating cardiomyocyte toxicity induced by DEHP, primary cardiomyocytes were divided into 4 groups (control group, DEHP group, TAX group and DEHP + TAX group). The results showed that in the cardiomyocytes, DEHP initiated apoptosis by increasing the expression of caspase-3, caspase-9, cyt c, and Bax at both the mRNA and protein levels and by decreasing the Bcl-2 levels compared with that of the control group. In addition, the activities of catalase (CAT), superoxide dismutase (SOD), and total antioxidative capacity (T-AOC) were clearly decreased (P < 0.05), while in the DEHP group, the malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels were observably increased (P < 0.05), compared with those in control group. Furthermore, compared with the control group, the DEHP group demonstrated a clear partial decrease in the expression of the mRNA levels of CYP1B1 and CYP2C18 (P < 0.05), and DEHP/TAX cotreatment partially prevented apoptosis and oxidative stress damage (P < 0.05). These results showed that exposure to DEHP induced apoptosis in chicken cardiomyocytes, while TAX could antagonize the toxicity of DEHP on cardiomyocytes by attenuating oxidative stress responses and modulating CYPs.

4-Bromodiphenyl Ether Causes Adrenal Gland Dysfunction in Rats during Puberty.

Polybrominated diphenyl ethers (PBDEs) are a group of flame retardants with two or more bromines attached. They are endocrine disruptors. PBDEs photodegrade into 4-bromodiphenyl ether (BDE3). Whether BDE3 impairs adrenal cortical cell function during postnatal development still remains unknown. The aim of the current study was to investigate the influence of BDE3 on adrenal cortical cell function. Sprague-Dawley rats (35 days of age, male) were orally administered with BDE3 (0, 50, 100, and 200 mg/kg/day body weight) for 21 days. BDE3 significantly increased serum aldosterone and corticosterone levels at 200 mg/kg without affecting adrenocorticotropic hormone level. Further study showed that BDE3 up-regulated Cyp11b1 at 100 and 200 mg/kg and Scarb1, Star, Cyp11b2, Cyp21, and Nr5a1 mRNA levels in the 200 mg/kg group. BDE3 also decreased the phosphorylation of AMP-activated protein kinase (AMPK) at 200 mg/kg and increased PGC-1α and phosphorylated cyclic AMP-responsive element-binding protein (CREB)/CREB at 200 mg/kg. Taken together, these findings demonstrate that BDE3 stimulates adrenal cell function likely through decreasing phosphorylation of AMPK and increasing phosphorylation of CREB.

AHR and GPER mediate the stimulatory effects induced by 3-methylcholanthrene in breast cancer cells and cancer-associated fibroblasts (CAFs)

BackgroundThe chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity.MethodsMolecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA.ResultsWe first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC.ConclusionsIn the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.

European Commission releases strategy addressing pharmaceuticals in the environment

Models of inflammation and infection, such as bacterial lipopolysaccharide (LPS), cause suppression of cytochrome P450 expression in various species, although the mechanisms involved are poorly understood. The effects of LPS...

SUN-044 Methylation Status and Gene Expression of Steroidogenic Enzymes in Benign Adrenocortical Tumors

DNA methylation has been recognized as a putative regulatory mechanism for CYP11B2 in primary hyperaldosteronism. We aimed to investigate the DNA methylation and the expression of a panel of genes encoding several enzymes involved in steroidogenesis in adrenocortical benign tumors. We collected a total of 60 adrenocortical tissues, including 9 non-functioning adrenal adenomas, 9 adenomas associated with autonomous cortisol secretion, 17 adenomas associated with Cushing’s syndrome, 13 Conn’s adenomas and 12 tissues derived from adrenal gland adjacent to the Conn’s adenomas. Non-functioning tumors and autonomous cortisol secretion were defined according to cortisol levels after 1 mg dexamethasone suppression test ≤ or > 50 nmol/L, respectively. The DNA methylation level of CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP21A2, DHCR24, HSD3B1, HSD3B2, NR5A1, STAR, and TSPO was evaluated by quantitative Bisulfite Next Generation Sequencing (bisulfite-NGS). Bioinformatic analysis was performed in a GalaxyProject environment and processed by BSPAT (Bisulfite Sequencing Pattern Analysis Tool). Spearman correlation coefficients were calculated using IBM SPSS 21 (IBM). CYP11B1, CYP11B2, CYP17, CYP21, STAR and β-actin gene expressions were examined by quantitative Real-Time PCR using a Sybr Green Assay kit (Thermo Fisher Scientific). The equation 2−ΔΔCt was used to calculate the fold changes in gene expression between the categories of samples. CYP11B2 was significantly hypomethylated in Conn’s adenoma, when compared to other adrenal tissues (P<0.001). No difference in methylation status was found among groups for the remaining genes. CYP11B2 mRNA levels were significantly higher in Conn’s adenoma than in the remaining adrenal tissues (P=0.001). CYP21 mRNA was significantly higher in all but Conn’s adenomas, when compared to normal adrenal tissues (P<0.001). Overall, we found a negative correlation between CYP11B2 expression and DNA methylation (rho= -0.379; P=0.003). DNA methylation seems to be a pivotal regulatory mechanism for CYP11B2 expression. It is feasible that epigenetic mechanisms may be responsible for aldosterone hypersecretion in Conn’s adenoma.

SUN-043 Epigenetic Regulation of Aldosterone Synthase Gene by Potassium

Purpose: DNA methylation is believed to be maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not stable. We reported that angiotensin II could change the DNA methylation status around transcription factor binding sites and a transcription start site (TSS) and activate expression of the aldosterone synthase gene (CYP11B2). In order to clarify the effect of potassium on the methylation status of CYP11B2 H295R cells were treated with high concentration of potassium and the methylation status of CYP11B2 was examined. Methods: H295R cells were treated with 16mM potassium for 7 days and observed the dynamic changes in DNA methylation patterns of the CYP11B2 promoter for 28 days. The gene expression of CYP11B2 was measured by real time quantitative PCR. Isolated DNAs from H295R cells were treated with bisulfite and amplified using primers specific for the human CYP11B2 promoter regions. Non-treated cells served as a control. Human CYP11B2 ELISA, chromatin accessibility by real-time PCR (CHART-PCR), DNA methylation and demethylation activity assay were done as previously reported. Results: DNA demethylation at CpG1 (Ad1) was detected within 2 days after stimulation. DNA at CpG2 (Ad5) and CpG3 and DNA around a transcription start site (CpG-1/-2) were demethylated at day 4 in cells treated with potassium. CYP11B2 mRNA levels in cells treated with potassium were significantly higher than those in non-treated cells at days 7 and 9. Similar changes were observed in the levels of CYP11B2 protein. CHART-PCR revealed that potassium treatment increased chromatin accessibility around Ad1 at days 2, 4, 7, 9 and 11, with accessibility increasing from 67% to 70%. Potassium treatment significantly reduced DNA methylation activity at days 7 and 9. Demethylation activity was not affected by potassium. Conclusion: Our data indicated that binding of transcription factors initiates chromatin relaxation and transcription, which are followed by DNA demethylation around transcription factor binding sites and a TSS in the CYP11B2 promoter. Decreased DNA methylation activity in the nucleus may play a role in DNA demethylation. DNA demethylation switches the phenotype of CYP11B2 expression from an inactive to an active state.

Glutamate affects the CYP1B1- and CYP2U1-mediated hydroxylation of arachidonic acid metabolism via astrocytic mGlu5 receptor.

The extrahepatic CYP enzymes, CYP1B1 and CYP2U1, have been predominantly found in both astrocytes and brain microvessels. We investigated the alteration in the production of hydroxyeicosatetraenoic acids (HETEs) from arachidonic acid (AA) mainly via CYP1B1 and CYP2U1 by glutamate. CYP1B1 and CYP2U1 mRNA levels were dose-dependently induced by glutamate in human U251 glioma cells and hCMEC/D3 blood-brain barrier cells. The increases in the CYP1B1 and CYP2U1 mRNA levels and the binding of CREB to CYP1B1 and CYP2U1 promoters following glutamate treatment were attenuated by mGlu5 receptor antagonist. The mRNA levels of CYP1B1 and CYP2U1 were increased in the cortex, hippocampus, and cerebellum from adult rats that received a subcutaneous injection of monosodium l-glutamate at 1, 3, 5, and 7 days of age; meanwhile, the protein levels of CYP1B1 and CYP2U1 in the astrocytes were induced by glutamate. Glutamate treatment significantly increased the production of 5-HETE, 8-HETE, 11-HETE, and 20-HETE in the cortex and cerebellum. These data suggested that the neuron-astrocyte reciprocal signaling can change the CYP-mediated AA metabolism (e.g. EETs and HETEs) in astrocytes via its specific receptor.

Evaluation of hepatotoxicity potential of a potent traditional Tibetan medicine Zuotai

Ethnopharmacological relevanceZuotai (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 studyTo evaluate the toxicological effects of Zuotai in hepatocytes and in zebrafish. Materials and methodsMTT 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. ResultsThe 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. ConclusionsResults showed that Zuotai induced hepatotoxicity effectively under a certain dose but its hepatotoxicity likely occurs via other mechanisms that did not depend on HgS.

Inflammatory breast cancer: Activation of the aryl hydrocarbon receptor and its target CYP1B1 correlates closely with Wnt5a/b-β-catenin signalling, the stem cell phenotype and disease progression

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.

Correction.

British Journal of PharmacologyVolume 175, Issue 22 p. 4281-4281 CORRECTIONFree Access Correction This article corrects the following: Schisandrol B protects against cholestatic liver injury through pregnane X receptors Hang Zeng, Yiming Jiang, Pan Chen, Xiaomei Fan, Dongshun Li, Aiming Liu, Xiaochao Ma, Wen Xie, Peiqing Liu, Frank J Gonzalez, Min Huang, Huichang Bi, Volume 174Issue 8British Journal of Pharmacology pages: 672-688 First Published online: March 16, 2017 First published: 02 October 2018 https://doi.org/10.1111/bph.14496AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat The authors of Zeng et al. (2017) have supplied the following correction to their article. In the Results subsection SolB up-regulates PXR genes involved in bile acid homeostasis, the text should say ‘SolB alone up-regulated hepatic levels of mRNAs for Cyp3a11, Cyp2b10, Ugt1a1, Oatp2 and Mrp3 (Figure 3A)’. Figure 3AOpen in figure viewerPowerPoint Due to the wrong primers for Cyp2b10 being used, the levels of Cyp2b10 mRNA were incorrectly observed. The authors recently realized this mistake and re-conducted the RT-qPCR assay using the correct primers, observing that SolB could induce hepatic Cyp2b10 mRNA levels in mice. Cyp2b10 is a target gene of PXR and takes part in bile acid detoxification, thus Cyp2b10 may also contribute to the anti-cholestatic effect of SolB. The authors would like to apologize for any inconvenience caused. The corrected Figure part is provided below: Reference Zeng H, Jiang Y, Chen P, Fan X, Li D, Liu A et al. (2017). Schisandrol B protects against cholestatic liver injury through pregnane X receptors. Brit J Pharmacol 174: 672– 688. https://doi.org/10.1111/bph.13729. Volume175, Issue22November 2018Pages 4281-4281 FiguresReferencesRelatedInformation

ATF6 knockdown decreases apoptosis, arrests the S phase of the cell cycle, and increases steroid hormone production in mouse granulosa cells.

Activating transcription factor 6 (ATF6), a sensor protein located in the endoplasmic reticulum (ER) membrane, is an important factor in the ER stress signaling pathway. ER stress is known to be involved in folliculogenesis, follicular growth, and ovulation; however, the physiological function of ATF6 in mouse granulosa cells remains largely unknown. The aim of this study was to assess the role of ATF6 in mouse granulosa cells with respect to apoptosis, the cell cycle, and steroid hormone production, as well as several key genes related to follicular development, via RNA interference, immunohistochemical staining, real-time quantitative PCR, Western blotting, flow cytometry, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay, and ELISA. Immunohistochemical staining revealed that ATF6 was extensively distributed in the granulosa cells of various ovarian follicles and oocytes in adult female mice. FSH or LH treatment significantly increased ATF6 protein levels in mouse granulosa cells. In the meantime, a recombinant plasmid was used to deplete ATF6 successfully using short hairpin RNA-mediated interference technology, which was verified at both the mRNA and protein levels. Flow cytometry and TUNEL assay analysis indicated that ATF6 depletion decreased apoptosis and arrested the S phase of the cell cycle in mouse granulosa cells. Consistent with these results, p53, caspase-3, B cell lymphoma 2 (Bcl-2)-associated X protein, CCAAT-enhancer-binding protein homologous protein, cyclin A1, cyclin B1, and cyclin D2 mRNA expression decreased, whereas Bcl-2 and glucose-regulated protein 78 kDa mRNA expression increased. Interestingly, ATF6 knockdown obviously increased progesterone and estradiol production in mouse granulosa cells. Cytochrome P450 1b1 (Cyp1b1) mRNA levels were downregulated, whereas Cyp11a1, steroidogenic acute regulatory, and Cyp19a1 mRNA levels were upregulated, in keeping with the changes in steroid hormones. Furthermore, ATF6 disruption remarkably increased insulin-like growth factor binding protein4 (Igfbp4) expression and decreased hyaluronan synthase 2 (Has2), prostaglandin-endoperoxide synthase 2 (Ptgs2), and prostaglandin F receptor (Ptgfr) expression in mouse granulosa cells, which are proteins crucial for follicular development. But, after treating with tunicamycin, the levels of Has2, Ptgs2, and Ptgfr increased relatively, whereas Igfbp4 expression decreased. Collectively, these results imply that ATF6, as a key player in ER stress signaling, may regulate apoptosis, the cell cycle, steroid hormone synthesis, and other modulators related to folliculogenesis in mouse granulosa cells, which may indirectly be involved in the development, ovulation, and atresia of ovarian follicles by affecting the physiological function of granulosa cells. The present study extends our understanding and provides new insights into the physiological significance of ATF6, a key signal transducer of ER stress, in ovarian granulosa cells.

Characteristics of Japanese aldosterone-producing adenomas with KCNJ5 mutations.

Somatic mutations in KCNJ5 gene have been identified in patients with adrenal aldosterone-producing adenomas (APAs). We previously reported that Japanese patients with APAs had distinct characteristics from patients in Western countries; i.e. they had a high frequency of KCNJ5 mutations and exhibited a frequent association with cortisol co-secretion. Therefore, APAs among Japanese patients may have different features from those in Western countries. We added recent cases, examined 47 cases (43% male) of APAs, including clinicopathological features, KCNJ5 mutations, and the mRNA levels of several steroidogenic enzymes, and compared the results obtained to those reported in other countries. While the prevalence of KCNJ5 mutations is approximately 40% in Western countries, 37 APA cases (78.7%) showed mutations: 26 with p.G151R and 11 with p.L168R. Although a significant gender difference has been reported in the frequency of KCNJ5 mutations in Europe, we did not find any gender difference. However, the phenotypes of Japanese patients with mutations were similar to those of patients in Western countries; patients were younger and had higher plasma aldosterone levels, lower potassium levels, and higher diastolic blood pressure. Reflecting these phenotypes, APAs with mutations had higher CYP11B2 mRNA levels. However, in contrast to APAs in Western countries, Japanese APAs with mutations showed lower CYP11B1, CYP17A1, and CYP11A1 mRNA levels. These findings demonstrated that Japanese APA patients may have distinct features including a higher prevalence of KCNJ5 mutations, no gender difference in the frequency of these mutations, and characteristics similar to the zona glomerulosa.

Evaluation of the human relevance of the constitutive androstane receptor-mediated mode of action for rat hepatocellular tumor formation by the synthetic pyrethroid momfluorothrin.

High dietary levels of the non-genotoxic synthetic pyrethroid momfluorothrin increased the incidence of hepatocellular tumors in male and female Wistar rats. Mechanistic studies have demonstrated that the mode of action (MOA) for momfluorothrin-induced hepatocellular tumors is constitutive androstane receptor (CAR)-mediated. In the present study, to evaluate the potential human carcinogenic risk of momfluorothrin, the effects of momfluorothrin (1-1,000 µM) and a major metabolite Z-CMCA (5-1,000 µM) on hepatocyte replicative DNA synthesis and CYP2B mRNA expression were examined in cultured rat and human hepatocyte preparations. The effect of sodium phenobarbital (NaPB), a prototypic rodent hepatocarcinogen with a CAR-mediated MOA, was also investigated. Human hepatocyte growth factor (hHGF) produced a concentration-dependent increase in replicative DNA synthesis in rat and human hepatocytes. However, while NaPB and momfluorothrin increased replicative DNA synthesis in rat hepatocytes, NaPB, momfluorothrin and Z-CMCA did not increase replicative DNA synthesis in human hepatocytes. NaPB, momfluorothrin and Z-CMCA increased CYP2B1/2 mRNA levels in rat hepatocytes. NaPB and momfluorothrin also increased CYP2B6 mRNA levels in human hepatocytes. Overall, while momfluorothrin and NaPB activated CAR in cultured human hepatocytes, neither chemical increased replicative DNA synthesis. Furthermore, to confirm whether the findings observed in vitro were also observed in vivo, a humanized chimeric mouse study was conducted. Replicative DNA synthesis was not increased in human hepatocytes of chimeric mice treated with momfluorothrin or its close structural analogue metofluthrin. As human hepatocytes are refractory to the mitogenic effects of momfluorothrin, in contrast to rat hepatocytes, the data support the hypothesis that the MOA for momfluorothrin-induced rat liver tumor formation is not relevant for humans.

Circadian Regulation of Benzo[a]Pyrene Metabolism and DNA Adduct Formation in Breast Cells and the Mouse Mammary Gland.

The circadian clock plays a role in many biologic processes, yet very little is known about its role in metabolism of drugs and carcinogens. The purpose of this study was to define the impact of circadian rhythms on benzo-a-pyrene (BaP) metabolism in the mouse mammary gland and develop a circadian in vitro model for investigating changes in BaP metabolism resulting from cross-talk between the molecular clock and aryl hydrocarbon receptor. Female 129sv mice (12 weeks old) received a single gavage dose of 50 mg/kg BaP at either noon or midnight, and mammary tissues were isolated 4 or 24 hours later. BaP-induced Cyp1a1 and Cyp1b1 mRNA levels were higher 4 hours after dosing at noon than at 4 hours after dosing at midnight, and this corresponded with parallel changes in Per gene expression. In our in vitro model, we dosed MCF10A mammary cells at different times after serum shock to study how time of day shifts drug metabolism in cells. Analysis of CYP1A1 and CYP1B1 gene expression showed the maximum enzyme-induced metabolism response 12 and 20 hours after shock, as determined by ethoxyresorufin-O-deethylase activity, metabolism of BaP, and formation of DNA-BaP adducts. The pattern of PER-, BMAL-, and aryl hydrocarbon receptor-induced P450 gene expression and BaP metabolism was similar to BaP-induced Cyp1A1 and Cyp1B1 and molecular clock gene expression in mouse mammary glands. These studies indicate time-of-day exposure influences BaP metabolism in mouse mammary glands and describe an in vitro model that can be used to investigate the circadian influence on the metabolism of carcinogens.

Activation of nuclear receptor CAR by an environmental pollutant perfluorooctanoic acid.

Perfluorocarboxylic acids (PFCAs) including perfluorooctanoic acid (PFOA) are environmental pollutants showing high accumulation, thermochemical stability and hepatocarcinogenicity. Peroxisome proliferator-activated receptor α is suggested to mediate their toxicities, but the precise mechanism remains unclear. Previous reports also imply a possible role of constitutive androstane receptor (CAR), a key transcription factor for the xenobiotic-induced expression of various genes involved in drug metabolism and disposition as well as hepatocarcinogenesis. Therefore, we have investigated whether PFCAs activate CAR. In wild-type but not Car-null mice, mRNA levels of Cyp2b10, a CAR target gene, were increased by PFOA treatment. PFCA treatment induced the nuclear translocation of CAR in mouse livers. Since CAR activators are divided into two types, ligand-type activators and phenobarbital-like indirect activators, we investigated whether PFCAs are CAR ligands or not using the cell-based reporter gene assay that can detect CAR ligands but not indirect activators. As results, neither PFCAs nor phenobarbital increased reporter activities. Interestingly, in mouse hepatocytes, pretreatment with the protein phosphatase inhibitor okadaic acid prevented an increase in Cyp2b10 mRNA levels induced by phenobarbital as reported, but not that by PFOA. Finally, in human hepatocyte-like HepaRG cells, PFOA treatment increased mRNA levels of CYP2B6, a CAR target gene, as did phenobarbital. Taken together, our present results suggest that PFCAs including PFOA are indirect activators of mouse and human CAR and that the mechanism might be different from that for phenobarbital. The results imply a role of CAR in the hepatotoxicity of PFCAs.

Effects of Cigarette Smoke Condensate on Oxidative Stress, Apoptotic Cell Death, and HIV Replication in Human Monocytic Cells.

While cigarette smoking is prevalent amongst HIV-infected patients, the effects of cigarette smoke constituents in cells of myeloid lineage are poorly known. Recently, we have shown that nicotine induces oxidative stress through cytochrome P450 (CYP) 2A6-mediated pathway in U937 monocytic cells. The present study was designed to examine the effect of cigarette smoke condensate (CSC), which contains majority of tobacco constituents, on oxidative stress, cytotoxicity, expression of CYP1A1, and/or HIV-1 replication in HIV-infected (U1) and uninfected U937 cells. The effects of CSC on induction of CYP1 enzymes in HIV-infected primary macrophages were also analyzed. The results showed that the CSC-mediated increase in production of reactive oxygen species (ROS) in U937 cells is dose- and time-dependent. Moreover, CSC treatment was found to induce cytotoxicity in U937 cells through the apoptotic pathway via activation of caspase-3. Importantly, pretreatment with vitamin C blocked the CSC-mediated production of ROS and induction of caspase-3 activity. In U1 cells, acute treatment of CSC increased ROS production at 6H (>2-fold) and both ROS (>2 fold) and HIV-1 replication (>3-fold) after chronic treatment. The CSC mediated effects were associated with robust induction in the expression of CYP1A1 mRNA upon acute CSC treatment of U937 and U1 cells (>20-fold), and upon chronic CSC treatment to U1 cells (>30-fold). In addition, the CYP1A1 induction in U937 cells was mediated through the aromatic hydrocarbon receptor pathway. Lastly, CSC, which is known to increase viral replication in primary macrophages, was also found to induce CYP1 enzymes in HIV-infected primary macrophages. While mRNA levels of both CYP1A1 and CYP1B1 were elevated following CSC treatment, only CYP1B1 protein levels were increased in HIV-infected primary macrophages. In conclusion, these results suggest a possible association between oxidative stress, CYP1 expression, and viral replication in CSC-treated cells of myeloid lineage. This study warrants a closer examination of the role of CYP1B1 in smoking-mediated enhanced HIV replication.

Synergistic and antagonistic interactions of binary mixtures of polycyclic aromatic hydrocarbons in the upregulation of CYP1 activity and mRNA levels in precision-cut rat liver slices

The current studies investigate whether synergistic or antagonistic interactions in the upregulation of CYP1 activity occur in binary mixtures of polycyclic aromatic hydrocarbons (PAHs) involving benzo[a]pyrene and five other structurally diverse PAHs of varying carcinogenic activity. Precision-cut rat liver slices were incubated with benzo[a]pyrene alone or in combination with a range of concentrations of a second PAH, and ethoxyresorufin O-deethylase, CYP1A1 and CYP1B1 mRNA levels determined. Concurrent incubation of benzo[a]pyrene with either dibenzo[a,h]anthracene or fluoranthene in liver slices led to a synergistic interaction, at least at low concentrations, in that ethoxyresorufin O-deethylase activity was statistically higher than the added effects when the slices were incubated with the individual compounds. In contrast, benzo[b]fluoranthene and, at high doses only, dibenzo[a,l]pyrene gave rise to antagonism, whereas 1-methylphenanthrene had no effect at all concentrations studied. When CYP1A1 mRNA levels were monitored, benzo[b]fluoranthene gave rise to an antagonistic response when incubated with benzo[a]pyrene, whereas all other compounds displayed synergism, with 1-methylphenathrene being the least effective. A similar picture emerged when CYP1B1 mRNA levels were determined, though the effects were less pronounced. In conclusion, it has been demonstrated that the benzo[a]pyrene-mediated upregulation of CYP1, at the mRNA and activity levels, is synergistically and antagonistically modulated by other PAHs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 764-775, 2017.

Comparative Expression Analysis of Cytochrome P450 1A1, Cytochrome P450 1B1 and Nuclear Receptors in the Female Genital and Colorectal Tissues of Human and Pigtailed Macaque.

This manuscript summarizes our recent progress in examine the CYP1A1 and CYP1B1 as well as a number of nuclear receptors in the female genital and colorectal tissues of human and pigtailed macaque. Understanding the nuclear receptor mediated regulation of CYP1A1 and 1B1 expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. However, there is a lack of systematic and comparative analysis of the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and clinically relevant animal models. The current study aims to fill this gap. We found CYP1A1, CYP1B1 and a number of nuclear receptors were expressed in the female genital and colorectal tissues of human and macaque. However, the mRNA level and protein localization of these CYP enzymes and NRs depended on the type of tissue examined. Cytochrome P450 (CYP) 1A1 and CYP1B1 activate hormonal and environmental procarcinogens, and are associated with carcinogenesis in female genital and colorectal tissues. Understanding the nuclear receptor (NR) mediated regulation of CYP expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. The study aims to analyze the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and pigtailed macaques. We found that compared to the liver, human CYP1A1 mRNA level in the genital and colorectal tissues was significantly lower, while the CYP1B1 level was significantly higher. CYP1A1 protein was mainly localized in the plasma membrane of the uterine and endocervical epithelial cells. The CYP1B1 protein was concentrated in the nucleus of genital and colorectal tissues. Fourteen NRs in the genital tract and 12 NRs in colorectal tissue were expressed at levels similar to or higher than the liver. The expression and localization of CYP1A1, CYP1B1, and NRs in macaque tissues were usually comparable to those of human tissues. In addition, menopause did not significantly alter the ectocervical mRNA levels of CYP1A1, CYP1B1, or NRs.

Nonsterol Isoprenoids Activate Human Constitutive Androstane Receptor in an Isoform-Selective Manner in Primary Cultured Mouse Hepatocytes.

Our laboratory previously reported that accumulation of nonsterol isoprenoids following treatment with the squalene synthase inhibitor, squalestatin 1 (SQ1) markedly induced cytochrome P450 (CYP)2B1 mRNA and reporter activity in primary cultured rat hepatocytes, which was dependent on activation of the constitutive androstane receptor (CAR). The objective of the current study was to evaluate whether isoprenoids likewise activate murine CAR (mCAR) or one or more isoforms of human CAR (hCAR) produced by alternative splicing (SPTV, hCAR2; APYLT, hCAR3). We found that SQ1 significantly induced Cyp2b10 mRNA (∼3.5-fold) in primary hepatocytes isolated from both CAR-wild-type and humanized CAR transgenic mice, whereas the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin had no effect. In the absence of CAR, basal Cyp2b10 mRNA levels were reduced by 28-fold and the effect of SQ1 on Cyp2b10 induction was attenuated. Cotransfection with an expression plasmid for hCAR1, but not hCAR2 or hCAR3, mediated SQ1-induced CYP2B1 and CYP2B6 reporter activation in hepatocytes isolated from CAR-knockout mice. This effect was also observed following treatment with the isoprenoid trans,trans-farnesol. The direct agonist CITCO increased interaction of hCAR1, hCAR2, and hCAR3 with steroid receptor coactivator-1. However, no significant effect on coactivator recruitment was observed with SQ1, suggesting an indirect activation mechanism. Further results from an in vitro ligand binding assay demonstrated that neither farnesol nor other isoprenoids are direct ligands for hCAR1. Collectively, our findings demonstrate that SQ1 activates CYP2B transcriptional responses through farnesol metabolism in an hCAR1-dependent manner. Further, this effect probably occurs through an indirect mechanism.

Abstract 40: Methylated resveratrol analogue DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cells

Abstract Resveratrol (3,4',5-trans-trihydroxystilbene) is a naturally occurring phytoalexin which shows a wide spectrum of biological activity. Although the anticancer effects of resveratrol have been revealed, its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. The structural alteration of the stilbene motif of resveratrol has been found to be a promising strategy for generation of several synthetic analogues with improved pharmacokinetic parameters. The substitution of hydroxyl groups of resveratrol for methoxy groups may increase the molecule stability, making it less susceptible to phase II conjugation reactions in vivo. The structure-activity studies have revealed that introduction of additional methoxy groups resulted in an increased cytotoxic properties of resveratrol analogues. Furthermore, the methoxy groups at positions 3,5- and 3,4,5- of the trihydroxystilbene scaffold have been identified as crucial for pro-apoptotic activity of the compound. To date few reports have demonstrated the antiproliferative effect of DMU-212 (3,4,4',5-tetramethoxystilbene) in transformed fibroblasts, prostate, liver, colon, cervical, breast and ovarian cancer cells. It has been shown that DMU-212 is biotransformed to four metabolites: 3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214), 4'-hydroxy-3,4,5-trimethoxystilbene (DMU-281), 4-hydroxy-3,5,4'-trimethoxystilbene (DMU-291), and 3-hydroxy-4,5,4'-trimethoxystilbene (DMU-807). However, if these metabolites retain the biological activity of DMU-212 remains unknown. Therefore, we evalueted the cytotoxic activity of these four metabolites of DMU-212 and the induction of apoptosis in ovarian A-2780 and SKOV-3 cancer cell lines. CYP1A1 and CYP1B1 are known to catalyze the conversion of estradiol to 4-hydroxyestradiol. Hence, the compounds such as resveratrol or DMU-212, which are structurally similar to the endogenous oestrogen estradiol, have been suggested to undergo metabolism by these enzymes. Surprisingly, DMU-212 has been shown to entirely inhibit CYP1B1 in ovarian A-2780 cancer cell line. Since CYP1B1 has been suggested to be involved in DMU-212 biotransformation, it can be hypothesised that one or more metabolites of DMU-212 regulate the expression of CYP1B1 by the feedback inhibition. To have an insight into DMU-212 metabolites' role in the expression of isoenzymes of CYP1 family we assessed CYP1A1 and CYP1B1 mRNA and protein levels in metabolites-treated A-2780 and SKOV-3 cancer cells. The inhibitory effect of four metabolites of DMU-212 was investigated in ovarian A-2780 and SKOV-3 cancer cell lines by MTT assays. The induction of apoptosis was assayed by Cell Death Detection ELISAPLUS test, Caspase-Glo® 3/7 luminescent test and flow cytometry analysis. The expression of CYP1A1 and CYP1B1 mRNA and protein levels was investigated by RT-qPCR and Western blott, respectively. From among four metabolites of DMU-212, DMU-214 exhibited the highest cytotoxic activity (IC50 A-2780=13.26±2.11 nM; IC50 SKOV-3=29.21±3.17nM). Furthermore, DMU-214 has been revealed to exert more pronounced inhibitory effects than the prototype compound, DMU-212. In addition, we observed that the stronger cytotoxic activity of DMU-214 evoked its greater pro-apoptotic effect. DMU-214 caused an increase in nucleosomes level in A-2780 and SKOV-3 cells, EF= 15.89 and 7.91, respectively. Exposure of either cell line to DMU-214 resulted in a statistically significant decrease in CYP1A1 and CYP1B1 mRNA and protein levels as compared to control. We showed for the first time that DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cell lines. Since DMU-214 has been observed to decrease expression of CYP1A1 and CYP1B1 in A-2780 and SKOV-3 cells our results provide a new insight into the concept of their feedback inhibition by the compound tested. Citation Format: Sylwia Borys, Malgorzata Kucinska, Dorota Trzybulska, Mariusz Kaczmarek, Marcin Wierzchowski, Marek Murias, Jadwiga Jodynis-Liebert, Hanna Piotrowska. Methylated resveratrol analogue DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 40.