Cytochrome P450 1A1 Gene Expression Research Articles

Fetal and neonatal dioxin exposure causes sex-specific metabolic alterations in mice.

Epidemiological studies report associations between early-life exposure to persistent organic pollutants (POPs) and impaired metabolic homeostasis in adulthood. We investigated the impact of early-life exposure to low-dose 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or 'dioxin') on the establishment of β-cell area during the perinatal period, as well as β-cell health and glucose homeostasis later in life. Adult female mice were injected with either corn oil (CO; vehicle control) or TCDD (20 ng/kg/day) 2×/week throughout mating, pregnancy, and lactation; offspring were thus indirectly exposed to maternal TCDD in utero and during lactation, with pollutant exposure ending at weaning. All offspring were maintained on chow diet from weaning until 12-17 weeks of age, after which a subset of CO- and TCDD-exposed offspring were transferred to a 45% high fat diet (HFD) as a metabolic stressor for an additional 10 weeks. TCDD significantly upregulated cytochrome P450 1a1 (Cyp1a1) gene expression in offspring pancreas at birth and weaning, indicating that maternal TCDD directly reaches the developing pancreas. TCDD-exposed pups were transiently hypoglycemic at birth and females were born with reduced % β-cell area, which persisted into adulthood. Early-life TCDD exposure had no persistent long-term effects on glucose homeostasis in chow-fed offspring, but when transferred to HFD, TCDD-exposed female offspring had a delayed onset of HFD-induced hyperglycemia, more pronounced HFD-induced hyperinsulinemia, and increase % PCNA+ β-cells compared with CO-exposed female offspring. This study demonstrates that early-life exposure of mice to TCDD has modest effects on metabolic health in chow-fed offspring but alters metabolic adaptability to HFD feeding in females.

Gefitinib Inhibits Rifampicin-Induced CYP3A4 Gene Expression in Human Hepatocytes.

During multidrug combination chemotherapy, activation of the nuclear receptor and the transcription factor human pregnane xenobiotic receptor (hPXR) has been shown to play a role in the development of chemoresistance. Mechanistically, this could occur due to the cancer drug activation of hPXR and the subsequent upregulation of hPXR target genes such as the drug metabolism enzyme, cytochrome P450 3A4 (CYP3A4). In the context of hPXR-mediated drug resistance, hPXR antagonists would be useful adjuncts to PXR-activating chemotherapy. However, there are currently no clinically approved hPXR antagonists in the market. Gefitinib (GEF), a tyrosine kinase inhibitor used for the treatment of advanced non-small-cell lung cancer and effectively used in combinational chemotherapy treatments, is a promising candidate owing to its hPXR ligand-like features. We, therefore, investigated whether GEF would act as an hPXR antagonist when combined with a known hPXR agonist, rifampicin (RIF). At therapeutically relevant concentrations, GEF successfully inhibited the RIF-induced upregulation of endogenous CYP3A4 gene expression in human primary hepatocytes and human hepatocells. Additionally, GEF inhibited the RIF induction of hPXR-mediated CYP3A4 promoter activity in HepG2 human liver carcinoma cells. The computational modeling of molecular docking predicted that GEF could bind to multiple sites on hPXR including the ligand-binding pocket, allowing for potential as a direct antagonist as well as an allosteric inhibitor. Indeed, GEF bound to the ligand-binding domain of the hPXR in cell-free assays, suggesting that GEF directly interacts with the hPXR. Taken together, our results suggest that GEF, at its clinically relevant therapeutic concentration, can antagonize the hPXR agonist-induced CYP3A4 gene expression in human hepatocytes. Thus, GEF could be a potential candidate for use in combinational chemotherapies to combat hPXR agonist-induced chemoresistance. Further studies are warranted to determine whether GEF has sufficient hPXR inhibitor abilities to overcome the hPXR agonist-induced chemoresistance.

Cytochrome P450 1A gene expression in response to therapies for the bacterial infection in fish.

Cytochrome P450 1A gene expression in response to therapies for the bacterial infection in fish.

In vivo and in vitro studies evaluating the chemopreventive effect of metformin on the aryl hydrocarbon receptor-mediated breast carcinogenesis.

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.

Commiphora myrrha (Nees) Engl. resin extracts induce phase-I cytochrome P450 2C8, 2C9, 2C19, and 3A4 isoenzyme expressions in human hepatocellular carcinoma (HepG2) cells

Commiphora myrrha (Nees) Engl. (C. myrrha) resin is the most Middle Eastern herbal medicine used against numerous diseases. After being decocted or macerated, this resin is widely consumed among Saudi Arabian patients who are already under prescribed medication. Despite its popularity, no studies have been reported on potential modulation effects of these resin extracts on drug metabolism. Therefore, we studied C. myrrha resin extracts on the expression of cytochrome P450 (CYP) drug-metabolizing isoenzyme in human hepatocellular carcinoma cell line HepG2. The C. myrrha extracts were prepared by sonication and boiling, resembling the most popular traditional preparations of maceration and decoction, respectively. Both boiled and sonicated aqueous extracts were fingerprinted using high-performance liquid chromatography equipped with ultra-violet detector (HPLC-UVD). The viability of HepG2 cells treated with these aqueous extracts was determined using CellTiter-Glo® assay in order to select the efficient and non-toxic resin extract concentrations for phase-I metabolic CYP isoenzyme expression analysis. The isoenzyme gene and protein expression levels of CYP 2C8, 2C9, 2C19, and 3A4 were assessed using reverse transcription-quantitative polymerase chain reaction and Western blot technologies. The HPLC-UVD fingerprinting revealed different chromatograms for C. myrrha boiled and sonicated aqueous extracts. Both aqueous extracts were toxic to HepG2 cells when tested at concentrations exceeding 150 µg/ml of the dry crude extract. The CYP 2C8, 2C9, and 2C19 mRNA expression levels increased up to 4.0-fold in HepG2 cells treated with either boiled or sonicated C. myrrha aqueous extracts tested between 1 and 30 µg/ml, as compared with the untreated cells. However, CYP3A4 mRNA expression level exceeded the 2.0-fold cutoff when the cells were exposed to 30 µg/ml of C. myrrha extracts. The up-regulation of CYP mRNA expression levels induced by both boiled and sonicated C. myrrha aqueous extracts was confirmed at the CYP protein expression levels. In conclusion, both sonicated and boiled C. myrrha aqueous extracts modulate CYP 2C8, 2C9, 2C19, and 3A4 gene expression at clinically-relevant concentrations regardless of preparation methods. Further in vitro and in vivo experiments are required for CYP isoenzyme activity assessment and the establishment of herb-drug interaction profile for these traditional medicinal resin extracts.

3′-Hydroxypterostilbene Inhibits 7,12-Dimethylbenz[a]anthracene (DMBA)/12-O-Tetradecanoylphorbol-13-Acetate (TPA)-Induced Mouse Skin Carcinogenesis

BackgroundA natural pterostilbene analogue isolated from the herb Sphaerophysa salsula, 3′-hydroxypterostilbene (HPSB), exhibits antiproliferative activity in several cancer cell lines; however, the inhibitory effects of HPSB on skin carcinogenesis remains unclear. PurposeThe aim of this study was to evaluate the inhibitory effects of HPSB on two-stage skin carcinogenesis in mice and its potential mechanism. Study Design and MethodsThis study investigated the anti-inflammatory and anti-tumor effects of HPSB in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated acute skin inflammation and 7,12-dimethylbenz[a]anthracene (DMBA)/TPA-induced two-stage skin carcinogenesis model. In addition, the effects of HPSB on the modulation of the phase I and phase II metabolizing enzymes in the DMBA-induced HaCaT cell model were investigated. ResultsThe results provide evidence that topical treatment with HPSB significantly inhibits TPA-induced epidermal hyperplasia and leukocyte infiltration through the down-regulation of cyclooxygenase-2 (COX-2), matrix metalloprotein-9 (MMP-9), and ornithine decarboxylase (ODC) protein expression in mouse skin. Furthermore, HPSB suppresses DMBA/TPA-induced skin tumor incidence and multiplicity via the inhibition of proliferating cell nuclear antigen (PCNA), Cyclin B1 and cyclin-dependent kinase 1 (CDK1) expression in the two-stage skin carcinogenesis model. In addition, pretreatment with HPSB markedly reduces DMBA-induced cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) gene expression in human keratinocytes; however, HPSB does not significantly affect the gene expression of the phase II enzymes. ConclusionThis is the first study to show that topical treatment with HPSB prevents mouse skin tumorigenesis. Overall, our study suggests that natural HPSB may serve as a novel chemopreventive agent capable of preventing carcinogen activation and inflammation-associated tumorigenesis.

Hypomethylation of the XRE -1383 site is associated with the upregulation of CYP1A1 in gastric adenocarcinoma.

Owing to its broad substrate specificity of mainly xenobiotics and its preferential extrahepatic expression, cytochrome P450 1A1 (CYP1A1) is a principle member of the CYP detoxifying enzyme superfamily involving in carcinogenesis. Methylation status of 93 CpG sites, densely scattered within approximately 1.5kb 5' regulatory region of CYP1A1, and its association with gene transcription was analyzed in tissue cohorts dissected from 40 patients with gastric cancer. Bisulfite sequencing and the resulting methylation percentages revealed dynamically methylated CpG sites located within or around xenobiotic response elements (XRE) 4-10, and a region of consistent hypermethylation located near proximal promoter, encompassing XRE2-3. Statistical analysis revealed significant differences of the methylation percentages at the CpG sites -1415 (0.032) and -1524 (P=0.041) (located at the close upstream region of XRE10) between cancerous and normal gastric tissues as well as between those with and without lymphatic involvement. Quantitative real time PCR analysis showed that the CYP1A1 gene expression significantly increases in cancerous tissues compared to their normal tissue cohort, and is significantly associated with hypomethylation at the CpG site -1383 (P=0.018) within the XRE10 motif. These data suggest that the variably methylated CpG site from the 5' regulatory region of CYP1A1, corresponding with the XRE10 regulatory region, is associated with its gene upregulation thus, is likely involved in gastric cancer incidence and metastasis. Methylation analysis of the CpG sites located within or around the XRE10 motif of the CYP1A1 promoter can be used as a potential marker to evaluate individual susceptibility to gastric cancer.

Sublethal effects of carbendazim in Jenynsia multidentata detected by a battery of molecular, biochemical and genetic biomarkers

The fungicide carbendazim (CBM) has been applied all around the world but its potential adverse effects other than its recognized activity as endocrine disruptor in non target organisms have been scarcely studied. The aims of this work were (1) to use a battery of biomarkers that can reflect potential negative effects such as oxidative stress, genotoxicity, neurotoxicity or altered immune response; and (2) to examine biomarkers of detoxification by analyzing the gene expression of cytochrome P4501A1 (CYP1A1) and the multi-xenobiotic resistance protein P-glycoprotein (P-gp) in the freshwater fish Jenynsia multidentata exposed to environmentally relevant concentrations of CBM during 24 h. Fish exposed to 5 μg/L showed inhibition of GST activity and an increase of TBARs contents in gills, the organ of direct contact with waterborne contaminants. Genotoxicity – measured in peripheral blood-was evidenced by the increases of micronuclei frequency when fish were exposed to 5, 10 and 100 μg/L CBM and of nuclear abnormalities (NA) frequency at 0.05, 0.5, 5, 10 and 100 μg/L CBM. The expression inhibition of interleukin (IL-1β) and tumor necrosis factor a (TNF-α) at 10, and 5 and 10 μg/L CBM, respectively, indicated an altered immune response. The expression of CYP1A1 was down regulated in liver at 10 μg/L and of P-gp at 5 μg/L CBM, indicating a possible slow on CBM metabolization. On the other hand, in gills CYP1A1 decreased at 5 and 10 μg/L while P-gp was induced at 5 and 100 μg/L CBM. Overall, most of these significant effects were detected below 10 μg/L CBM, in a range of realistic concentrations in aquatic ecosystems worldwide.

Dissociation of the AhR/ARNT complex by TGF-β/Smad signaling represses CYP1A1 gene expression and inhibits benze[a]pyrene-mediated cytotoxicity

Cytochrome P450 1A1 (CYP1A1) catalyzes the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) and is transcriptionally regulated by the aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) complex upon exposure to PAHs. Accordingly, inhibition of CYP1A1 expression reduces production of carcinogens from PAHs. Although transcription of the CYP1A1 gene is known to be repressed by transforming growth factor-β (TGF-β), how TGF-β signaling is involved in the suppression of CYP1A1 gene expression has yet to be clarified. In this study, using mammalian cell lines, along with shRNA-mediated gene silencing, CRISPR/Cas9-based genome editing, and reporter gene and quantitative RT-PCR assays, we found that TGF-β signaling dissociates the B[a]P-mediated AhR/ARNT heteromeric complex. Among the examined Smads, Smad family member 3 (Smad3) strongly interacted with both AhR and ARNT via its MH2 domain. Moreover, hypoxia-inducible factor 1α (HIF-1α), which is stabilized upon TGF-β stimulation, also inhibited AhR/ARNT complex formation in the presence of B[a]P. Thus, TGF-β signaling negatively regulated the transcription of the CYP1A1 gene in at least two different ways. Of note, TGF-β abrogated DNA damage in B[a]P-exposed cells. We therefore conclude that TGF-β may protect cells against carcinogenesis because it inhibits CYP1A1-mediated metabolic activation of PAHs as part of its anti-tumorigenic activities.

Gefitinib, at Its Clinically Relevant Concentrations, Inhibits Rifampicin‐Induced CYP3A4 Gene Expression in Human Hepatocytes

Pregnane xenobiotic receptor (PXR) has been shown to induce chemoresistance during multidrug chemotherapy via upregulation of PXR target genes such as cytochrome P450 3A4 (CYP3A4). CYP3A4 contributes to the metabolism of over 50% of clinical drugs. Therefore, during multidrug chemotherapy, drug induction of hPXR‐mediated CYP3A4, can reduce the therapeutic concentration of coadministered drugs, leading to chemoresistance. It is possible to overcome a PXR agonist‐induced chemoresistance with a PXR antagonist. However, proposed antagonists lack appropriate pharmacological characteristics that allow them to be active in the clinic. We propose that a PXR antagonist would be a cancer drug itself that is part of a “cancer drug cocktail”, and effective as PXR antagonist at therapeutic concentrations. Gefitinib (GEF), a tyrosine kinase inhibitor approved for the treatment of advanced non‐small cell lung cancer, and shown to be effective in combinational chemotherapy treatment, is a promising candidate due to its PXR ligand‐like features. We therefore sought to determine whether GEF would behave as a PXR antagonist at its clinically relevant concentrations in human primary hepatocytes, human hepatocells, and HepG2 human hepatocellular carcinoma cells. Rifampicin, an agonist of PXR, was used to activate PXR‐mediated CYP3A expression. Reporter gene assays were conducted to determine PXR transactivation of CYP3A4 promoter activity. Cell viability and qRT‐PCR assays were performed to assess the cytotoxicity and CYP3A4 gene expression, respectively. PXR molecular docking studies were carried out to predict binding affinity of GEF. Finally, competitive ligand binding and steroid receptor coactivator‐1 (SRC‐1) recruitment assays were performed to examine GEF ability to bind to PXR and alter SRC‐1 recruitment. GEF, at its therapeutic concentrations, repressed rifampicin‐induced endogenous CYP3A4 gene expression. Additionally, GEF inhibited rifampicin induction of PXR‐mediated CYP3A4 promoter activity. These findings indicate that GEF could inhibit PXR agonists‐induced CYP3A4 gene expression. Molecular docking studies predicted that GEF can bind to multiple sites on PXR, including the ligand binding pocket, AF‐2 region and alpha‐8 pocket. Indeed, GEF bound to PXR and attenuated the PXR agonist‐induced SRC‐1 interaction, suggesting that GEF directly interacts with multiple sites on the PXR. Taken together, these results suggest that GEF, at its clinically relevant therapeutic concentration, can antagonize PXR agonists‐induced CYP3A4 gene expression. Thus, GEF could be a potential candidate for use in combinational chemotherapies to combat PXR‐mediated chemoresistance. Future studies are required to examine GEF selectivity towards PXR, and to determine GEF suppression of PXR agonists‐induced chemoresistance.Support or Funding InformationThe authors thank Drs. Coleman, Mansour, Schwartz & Tao for sharing their research facilities. This work was supported by the Auburn University Research Initiative in Cancer Grant and Animal Health and Disease Research Grant.

Analysis of Streptococcus agalactiae ‐induced liver injury in tilapia ( Oreochromis niloticus )

Streptococcosis, a bacterial disease for multiple fish species, especially tilapia, caused more disruption to the fish culture industry. However, the underlying mechanisms for the occurrence of streptococcosis remain unclear. The aim of this study was to study the effects of streptococcus on the liver of tilapia. In this study, tilapia were injected streptococcal solution (0.05 ml/10 g body weight) and collected blood and liver at 4, 8, 24, 48, 72, 96, 120 and 144 hr after injection. The results showed that the activities of glutamate oxalate transaminase (GOT), glutamate pyruvate transaminase (GPT), alkaline phosphatase (AKP) and superoxide dismutase (SOD) in the serum of tilapia exhibited irregular changes: increase at first, then decrease and increase again. The levels of total antioxidant capacity (T-AOC), glutathione (GSH) and malondialdehyde (MDA) showed a decreasing tendency in serum after injection, except GSH at 4 hr. The activity of catalase (CAT) decreased in serum at 4, 24, 120 and 144 hr after injection of streptococcal solution, while the content TP enhanced in serum at 48, 72 and 120 hr after injection. Observations of pathological sections showed obvious damage to the liver tissue structure in response to streptococcal infection. Western blotting revealed an increase in ikkbeta (Ikkβ) protein with prolonged infection time and a decrease in NF-kappa-B inhibitor alpha (IkB-α) and ikappab kinase alpha (Ikkα) proteins. The gene expression of cytochrome P4501A (CYP1A) was downregulated in liver at 24 and 48 after injection, while the mRNA levels of heat stress protein 70 (HSP70) and tumour necrosis factor alpha (TNF-α) increased at 48 and/or 72 hr. Taken together, our findings confirmed that streptococcus can cause serious damage to the liver of tilapia.

Selective cytochrome P450 1A1 but not 1B1 promoterCpG island DNA methylation by 6-formylindolo[3,2-b]carbazole (FICZ).

Epigenetic alterations are essential for normal mammalian development and regulation of gene expression. In this study, we aimed to determine if an enigmatic endogenous ligand of the aryl hydrocarbon receptor (AHR), 6-formylindolo[3,2-b]carbazole (FICZ), and methionine (Meth) have an epigenetic impact on AHR-regulated cytochrome P450 1A1 and B1 (CYP1A1 and CYP1B1) gene expression. Human hepatoma (HepG2-XRE-Luc and huh7) cells were exposed to FICZ in a medium with and without Meth supplementation. Selective and transient silencing of CYP1A1 but not CYP1B1 were seen by FICZ. Here we found that FICZ transiently represses CYP1A1 by targeting DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and concomitant DNA methylation of the CYP1A1 promoter gene. Treatments with 5-aza-dC augmented CYP1A1 transcription activity. Our results reveal a new mechanism for transient activation of AHR by FICZ that can negatively and positively influence gene expression, and highlight the regulatory role of Meth on the CYP1A1 gene expression.

P3493Uremic toxin induced endothelial dysfunction can be restored by inhibition of the aryl hydrocarbon receptor: implications for cardiovascular disease treatment in chronic kidney disease patients

Abstract Introduction Cardiovascular disease (CVD) mortality is significantly higher in chronic kidney disease (CKD) patients, with vascular calcification and atherosclerosis proving to be major contributors. Endothelial dysfunction is an early marker of atherosclerosis, triggered by oxidative stress and reduced nitric oxide production. The uremic toxin indoxyl sulphate (IS), a protein-bound non-dialyzable molecule derived from dietary tryptophan that accumulates in the blood of CKD patients, activates the aryl hydrocarbon receptor (AhR) promoting downstream cytochrome P450 1A1 (CYP1A1) expression mediating oxidative stress and potentially endothelial dysfunction. Targeting IS-induced AhR activation in the endothelium has not previously been examined and may represent a novel approach in targeting endothelial dysfunction. Purpose To assess the ability of the AhR antagonist, CH223191, to inhibit IS-mediated impairment of endothelial function and disruption of redox balance. Methods To assess endothelium-dependent relaxation, the thoracic aorta from adult male Sprague Dawley rats (N=10) were used in ex vivo experiments. Rings (5mm) from the aorta were exposed to IS (300μM) in the presence and absence of the AhR antagonist CH223191 (1 and 10μM), prior to pre-constriction of the vessel with phenylephrine (30μM) followed by relaxation with acetylcholine (ACh; 1nM-30μM). Control rings were not exposed to IS or CH223191 to determine normal endothelial responses to ACh. Responses were recorded with isometric force transducers connected to a PowerLab using Chart Software. Tissue sections from these rings were stained for superoxide using dihydroethidium. To examine key AhR-mediated oxidative stress pathways, separate aortic rings were exposed to IS and CH223191, under the above conditions, for 4 hours prior to RNA extraction and real-time PCR analysis. Results IS reduced the maximum level of endothelium-dependent relaxation (Rmax) to 50.17±2.71% (P<0.001) compared to control (86.00±3.93%). In the presence of IS, CH223191 restored Rmax to 77.74±3.14% (1μM) and 81.63±3.27% (10μM) (Figure, P<0.001). The potency of ACh, known as the pEC50 (negative logarithm of the effective concentration of ACh to produce a relaxation response of 50%), in control tissues (−7.08±0.07) was increased 100-fold following exposure of IS (−5.10±0.13; P<0.001). CH223191 restored pEC50 back to control values (1μM, −6.62±0.09; 10μM, −6.83±0.08; P<0.05). IS-exposed rings increased superoxide expression (P<0.001) and CYP1A1 gene expression (P<0.001), CH223191 restored expression of both superoxide (P<0.001) and CYP1A1 (P<0.001) back to control levels. CH223191 restores endothelial function Conclusion Our findings demonstrate the adverse impact of IS-mediated AhR activation on the vascular endothelium, where oxidative stress may play a critical role inducing endothelial dysfunction in the vasculature of the heart and kidneys. AhR inhibition may provide a novel therapy for CVD in the CKD setting. Acknowledgement/Funding National Health and Medical Research Council of Australia Program Grant

Primary endometrial 3D co-cultures: A comparison between human and rat endometrium

Human and rat reproductive systems differ significantly with respect to hormonal cyclicity and endometrial cell behavior. However, species-differences in endometrial cell responses upon hormonal stimulation and exposure to potentially toxic compounds are poorly characterized. In this study, human and rat endometrial hormonal responses were assessed in vitro using a 3D co-culture model of primary human and rat endometrial cells. The models were exposed to the aryl hydrocarbon receptor (AHR) ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), laquinimod, and its AHR active metabolite DELAQ.In both the human and rat endometrial models, estrogen receptor and progesterone receptor gene expression was modulated by the hormonal treatments, comparable to the in vivo situation. AHR gene expression in the human endometrial model did not change when exposed to hormones. In contrast, AHR expression decreased 2-fold in the rat model when exposed to predominantly progesterone, which resulted in a 2.8-fold attenuation of gene expression induction of cytochrome P450 1A1 (CYP1A1) by TCDD. TCDD and DELAQ, but not laquinimod, concentration-dependently induced CYP1A1 gene expression in both human and rat endometrial models. Interestingly, the relative degree of DELAQ to induce CYP1A1 was higher than that of TCDD in the human model, while it was lower in the rat model.These data clearly show species-differences in response to hormones and AHR ligands between human and rat endometrial cells in vitro, which might greatly affect the applicability of the rat as translational model for human endometrial effects. This warrants further development of human relevant, endometrium-specific test methods for risk assessment purposes.

Peppermint (Mentha piperita L.) extract effectively inhibits cytochrome P450 3A4 (CYP3A4) mRNA induction in rifampicin-treated HepG2 cells.

Interaction between foods and drugs is an important consideration in pharmaceutical therapy. Therefore, here, we examined the suppressive effects of the extracts from seven edible herbs on the induction of CYP3A4 gene expression in rifampicin-treated HepG2 cells. We evaluated the structure and suppressive activity of the most effective active compound isolated from dried peppermint (Mentha piperita L.). The structure of the compound was identified as that of pheophorbide a based on spectroscopic data. It suppressed the induction of CYP3A4 mRNA expression by rifampicin in a dose-dependent manner. Quantitative high-performance liquid chromatography showed that 2 g of dry leaves 0.43 mg in one cup of peppermint tea. These findings demonstrate that pheophorbide a suppresses the induction of CYP3A4 mRNA expression in rifampicin-treated HepG2 cells. Pheophorbide is known to cause photosensitivity. However, the effective dose of pheophorbide a that had a suppressive effect was very low, indicating a high safety margin. Abbreviations: DAD: diode array detector; DMEM: Dulbecco's modified Eagle's medium; ELISA: enzyme-linked immunosorbent assay; HPLC: high-performance liquid chromatography; PCR: polymerase chain reaction; PXR: pregnane X receptor; CAR: constitutive androstane receptor; AHR: aryl hydrocarbon receptor; TLC: thin-layer chromatography.

Anti-tumor properties of methoxylated analogues of resveratrol in malignant MCF-7 but not in non-tumorigenic MCF-10A mammary epithelial cell lines

Resveratrol is a plant-derived polyphenol that is known for its anti-inflammatory and anti-tumorigenic properties in in vitro and in vivo models. Recent studies show that some resveratrol analogues might be more potent anti-tumor agents, which may partly be attributed to their ability to activate the aryl hydrocarbon receptor (AHR). Here, the anti-tumorigenic properties of resveratrol and structural analogues oxyresveratrol, pinostilbene, pterostilbene and tetramethoxystilbene (TMS) were studied in vitro, using in the malignant human MCF-7 breast cancer cell line and non-tumorigenic breast epithelial cell line MCF-10A.Cell viability and migration assays showed that methoxylated analogues of resveratrol are more potent anti-tumorigenic compounds than resveratrol and its hydroxylated analogue oxyresveratrol, with 2,3’,4,5’-tetramethoxy-trans-stilbene (TMS) being the most potent compound. TMS decreased MCF-7 tumor cell viability with 50% at 3.6 μM and inhibited migration with 37.5 ± 14.8% at 3 μM. In addition, TMS activated the AHR more potently (EC50 in a reporter gene assay 2.0 μM) and induced AHR-mediated induction of cytochrome P450 1A1 (CYP1A1) activity (EC50 value of 0.7 μM) more than resveratrol and the other analogues tested. Cell cycle analysis showed that TMS induced a shift in cell cycle status from the G1 to the G2/M phase causing a cell cycle arrest in the MCF-7 cells, while no effect of TMS was observed in the non-tumorigenic MCF-10A mammary epithelial cell line. Gene expression analysis showed that 3 μM TMS increased gene expression of CYP1A1 (289-fold), CYP1B1 (5-fold) and Nqo1 (2-fold), and decreased gene expression of IL-8 (3-fold) in MCF-7 cells. In MCF-10A cells, 10 μM TMS also increased gene expression of CYP1A1 (5-fold) and CYP1B1 (2-fold), but decreased gene expression of Nqo1 (1.4-fold) in contrast to MCF-7 cells.TMS displays more potent anti-tumorigenic properties and activates the AHR more effectively than resveratrol. In addition, this is the first study to show that TMS, but not resveratrol, selectively inhibits the cell cycle of breast tumor cells and not the non-tumorigenic cells. Our study provides more insight in the anti-tumor properties of the methoxylated analogues of resveratrol in breast cells in vitro.

Gancao (Glycyrrhizae Radix) provides the main contribution to Shaoyao-Gancao decoction on enhancements of CYP3A4 and MDR1 expression via pregnane X receptor pathway in vitro

BackgroundChinese herbal formula Shaoyao Gancao decoction (SGD) is often used as an adjuvant with chemotherapeutic agents to treat cancer. Due to the herb-drug interactions, the alternations of drug metabolic enzyme and drug transporters induced by SGD deserve to be explored. We aimed to investigate the effect of SGD on the pregnane X receptor (PXR)-mediated transcriptional regulation of cytochrome P450 3A4 (CYP3A4) and drug transporter multidrug resistance protein 1 (MDR1) in vitro. Besides, we assessed the contribution of constituent herbs to SGD on the regulation of CYP3A4 and MDR1.MethodsThe dual luciferase reporter gene system containing the hPXR expression plasmid and the reporter gene plasmid of CYP3A4 or MDR1 was co-transfected to HepG2 and Caco2 cells. Luciferase activities were determined using a Dual-luciferase reporter assay kit. The gene expression of CYP3A4 and MDR1 in the hPXR-transfected LS174T cells were assessed by real-time qPCR. Finally, the contribution of constituent herbs from SGD was evaluated.ResultsSGD, Shaoyao and Gancao concentration-dependently increased promoter activities of CYP3A4 and MDR1 in vitro. Moreover, SGD, Shaoyao and Gancao up-regulated CYP3A4 and MDR1 mRNA in hPXR-transfected LS174T cells. As the herbal constituent of SGD, Gancao possesses significantly higher levels of metabolic enzyme and drug transporters compared with Shaoyao.ConclusionSGD tends to enhance CYP3A4 and MDR1 expression via PXR pathway, especially Gancao provides the main contribution. This study highlights a potential in vitro mechanism for SGD on the regulation of drug metabolic enzymes and drug transporters.

Benzo(a)pyrene suppresses tracheal antimicrobial peptide gene expression in bovine tracheal epithelial cells

Respiratory disease is an important cause of morbidity and mortality in cetaceans, which are also threatened by environmental degradation caused by crude oil spills. Following oil spills, cetaceans at the water surface may inhale droplets of oil containing toxic polycyclic aromatic hydrocarbons (PAHs), which could potentially alter respiratory immunity via activation of the aryl hydrocarbon receptor (AHR) and its subsequent interaction with nuclear factor kappa B (NF-κB). β-defensins are antimicrobial peptides secreted by airway epithelial cells and their expression is known to be dependent on NF-κB. We hypothesized that PAHs may suppress the expression of β-defensins, and thereby contribute to the pathogenesis of pneumonia. This hypothesis was modeled by measuring the in vitro effects of benzo(a)pyrene (BAP), phenanthrene, and naphthalene on tracheal antimicrobial peptide (TAP) gene expression in bovine tracheal epithelial cells. Stimulation with lipopolysaccharide (LPS) induced 20 ± 17-fold (mean ± SD) increased TAP gene expression. Exposure of tracheal epithelial cells to 5 μM BAP for 4 or 8 h, followed by incubation with a combination of LPS and 5 μM BAP for another 16 h, significantly (P = 0.002) suppressed LPS-induced TAP gene expression by 40.6 ± 21.8% (mean ± SD) in tracheal epithelial cells from 9 calves tested. BAP-induced suppression of TAP gene expression coincided with induction of cytochrome P450 1A1 gene expression. In contrast, phenanthrene and naphthalene had no consistent effect, and exposure to PAHs did not significantly affect constitutive TAP gene expression (i.e. without LPS). These findings characterize the suppressive effects of BAP—a toxic pollutant found in crude oil—on this respiratory innate immune response.

Novel plant inducers of PXR-dependent cytochrome P450 3A4 expression in HepG2 cells

The cytochrome P450 3A4 (CYP3A4) is the most abundant CYP450 enzyme involved in the metabolism of endogenous products and xenobiotics, including prescription drugs and herbals. Modulation of hepatic CYP3A4 gene expression via nuclear receptors, like pregnane X receptor (PXR), is a major cause of adverse effects like drug-unresponsiveness and toxicity. In the present study, ethanol extracts of 58 medicinal plants, belonging to 27 families, were evaluated for potential activities in CYP3A4 induction in HepG2 cells by reporter gene assay. For PXR-mediated CYP3A4 induction, a 50 μg/ml concentration was used for all non-cytotoxic plants extracts. Rifampicin (10 μM) and DMSO (0.1%) were used as standard inducer and untreated (negative) control, respectively. The comparative fold-induction of CYP34A by the plant extracts in relation to the untreated control was determined. As a result, Dodonaea angustifolia (2.62 fold; P < 0.0001) was found to be the most promising inducer of CYP3A4, followed by Euphorbia tirucalli (1.95 fold; P = 0.0004), Alternanthera pungens (1.74 fold, P = 0.0035), and Ficus palmata (1.65 fold; P = 0.0097). Further phytochemical characterizations of the active plants are therefore, warranted.

Belinostat Inhibits Rifampicin Induced CYP3A4 and MDR1 Gene Expression

Activation of pregnane X receptor (PXR) by clinical compounds during multidrug chemotherapy results in upregulation of the expression of PXR target genes, including cytochrome p450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1), leading to chemoresistance. It is possible to overcome the PXR‐mediated chemoresistance by downregulating the upregulated PXR target genes by inhibiting the activated PXR. However, a selective and less‐toxic PXR antagonist has yet to be developed. In this regard, a clinical anticancer drug, with selective PXR antagonistic activity at its less‐toxic concentrations, would be beneficial. We sought to determine whether belinostat, a clinically‐used histone deacetylase inhibitor, inhibits the PXR target gene expression at its clinically relevant plasma concentrations (&lt; ~100 μM) in human hepatocytes (primary hepatocytes &amp; hepatocells) and intestinal cells (LS174T colon cancer cells). Rifampicin, an agonist of human PXR, was used to activate PXR. Cell viability and CYQUANT cell proliferation assays were performed to determine cytotoxicity and cell proliferation, respectively. Quantitative RT‐PCR assays were conducted to study the gene expression. CYP3A4 p450‐Glo and Rhodamine‐123 intracellular accumulation assays were performed to determine the function of CYP3A4 and MDR1, respectively. Belinostat, at its unbound therapeutic plasma concentrations (&lt; ~5 μM) did not affect the viability of LS174T cells and the hepatocytes. Belinostat (1 &amp; 3 μM) not only inhibited rifampicin‐induced gene expression of CYP3A4 and MDR1, but also attenuated rifampicin‐induced activity of CYP3A4 and MDR1. However, belinostat alone did not affect CYP3A4 or MDR1 gene expression. These results suggest that belinostat does not affect the basal expression of PXR target genes but downregulates the upregulated PXR target genes by inhibiting the ligand‐activated PXR. Notably, belinostat, at its PXR inhibiting concentrations, decreased rifampicin‐induced proliferation of LS174T cells, suggesting that belinostat suppresses PXR‐mediated proliferation of the cancer cells. Interestingly, belinostat failed to inhibit rodent PXR agonist pregnenolone‐16 alpha‐carbonitrile (PCN)‐induced expression of CYP3A1 (the rat analog of human CYP3A4) in rat primary hepatocytes, suggesting that belinostat exhibits species‐specific inhibition of PXR at unbound plasma therapeutic concentrations. Taken together, these results are consistent with the conclusion that belinostat, at its less‐toxic and clinically relevant unbound plasma concentrations, inhibits the ligand‐activated human PXR target gene expression. Future studies will determine the mechanisms of belinostat inhibition of PXR, and belinostat sensitization of the cancer cells to chemotherapy drugs with PXR agonistic activity.Support or Funding InformationThe authors would like to thank Drs. Coleman, Schwartz, and Tao for sharing their research facilities. This work was supported by the Auburn University Research Initiative in Cancer Grant, Animal Health and Disease Research Grant, and Auburn University Startup Funds to Pondugula SR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.