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Chemico-biological Interactions Sep 2022Polygonum multiflorum Thunb. (PMT) is the most common traditional Chinese medicine used to treat multiple diseases, and the hepatotoxicity caused by PMT has made great...
BACKGROUND & AIMS
Polygonum multiflorum Thunb. (PMT) is the most common traditional Chinese medicine used to treat multiple diseases, and the hepatotoxicity caused by PMT has made great concern around world. Recent results showed that emodin is the potential toxic components of PMT, but the molecular mechanisms of emodin on liver toxicity remain to be elucidated.
METHODS
Evaluation of parent- and metabolite-induced cytotoxicity in emodin were compared in L02 cells and mouse model from the perspective of drug metabolizing enzymes. The effect and mechanism of emodin-induced hepatotoxicity were analyzed using electrophoretic mobility shift, promoter reporter, and high content screening.
RESULTS
We showed that emodin treatment (360 mg/kg in mice, 50 μM in L02 cells) induced hepatotoxicity and enhanced reactive oxidative stress (ROS) level. Importantly, emodin-induced ROS accumulation and hepatotoxicity were attenuated in the condition of CH223191, a selective inhibitor of aryl hydrocarbon receptor (AhR), and aggravated by 3-methylcholanthrene, a selective activator of AhR. Interestingly, we performed the study on ROS mediated ER stress and mitochondrial dysfunction in emodin-induced hepatotoxicity, the results showed that emodin can decrease MMP and trigger ER stress with Ca overloading and the expression of ATF4 increasing, further resulted with increased apoptosis in L02 cells and mice mortality rate, while the changes were alleviated by CH223191. Furthermore, the 5-hydroxyemodin, a metabolite by emodin through CYP1A2 enzyme, showed more severe hepatotoxicity compared to emodin.
CONCLUSIONS
Our results validated that the metabolism of emodin to 5-hydroxyemodin by CYP1A played an important role in the hepatocellular toxicity of emodin and provided evidence that CYP1A1 and AhR could be used to predict and validate patient-specific liver injury of PMT or other herbs containing emodin.
Topics: Animals; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP1A1; Emodin; Methylcholanthrene; Mice; Reactive Oxygen Species; Receptors, Aryl Hydrocarbon
PubMed: 35934134
DOI: 10.1016/j.cbi.2022.110089 -
Toxicology Jan 2020We have previously shown that daily exposure to the environmental pollutant 3-methylcholanthrene (3MC) alters the ovarian function by affecting follicle growth and... (Comparative Study)
Comparative Study
We have previously shown that daily exposure to the environmental pollutant 3-methylcholanthrene (3MC) alters the ovarian function by affecting follicle growth and ovulation. To extend our findings, the aims of this work were to study the effects of daily and non-daily exposure to 3MC on oocyte morphology and integrity and the meiosis process. To this end, immature female rats were daily (0.1-1.0 mg/kg) and non-daily (0.1 mg/kg, three times a week) exposed to 3MC and/or α-naphthoflavone (αNF) (80 mg/kg) for 19 and 20 days, respectively. The latter was used to study its ability to prevent the 3MC action. Follicular growth was examined by histology, apoptosis by in situ cell death detection, oocyte integrity by morphological parameters and fluorescent dyes, and the meiotic spindle by immunostaining. Compared with controls (C), and in a dose-dependent manner, all 3MC-treated rats showed i) increased presence of apoptotic cells in antral follicles and decreased percentage of healthy oocytes, ii) increased oocyte area, perimeter and perivitelline space and decreased thickness of the zona pellucida, and ii) increased percentage of oocytes with abnormal meiotic spindle. In addition, the non-daily dose of 3MC caused DNA damage in oocytes, but not in blood or bone marrow cells. All 3MC-induced changes were prevented with the co-treatment with αNF. These results suggest that low doses of 3MC severely disrupt the ovarian function and that germ cells seem to be more sensitive to this environmental pollutant than other cells such as peripheral blood and bone marrow cells.
Topics: Animals; Apoptosis; Benzoflavones; Bone Marrow Cells; DNA Damage; Dose-Response Relationship, Drug; Environmental Pollutants; Female; Meiosis; Methylcholanthrene; Oocytes; Ovarian Follicle; Rats; Rats, Sprague-Dawley
PubMed: 31712135
DOI: 10.1016/j.tox.2019.152328 -
Acta Physiologica (Oxford, England) Apr 2016The aim of this work was to identify the role of the NADPH oxidase Nox4 for tumour angiogenesis in a slow-growing tumour model in mice.
AIM
The aim of this work was to identify the role of the NADPH oxidase Nox4 for tumour angiogenesis in a slow-growing tumour model in mice.
METHODS
Tumour angiogenesis was studied in tumours induced by the carcinogen 3-methylcholanthrene (MCA) in wild-type and Nox knockout mice. Mice were killed when the tumour reached a diameter of 1.5 cm and tumour tissue was used for histological and molecular analysis.
RESULTS
3-methylcholanthrene induced fibrosarcoma in wild-type, Nox1y/-, Nox2y/- and Nox4-/- mice. Histological analysis of vessel density using anti-CD31 staining showed a significant 38% reduction in tumour vascularization in fibrosarcomas of Nox4-/- mice. In contrast, tumour angiogenesis was doubled in Nox1 knockout mice, whereas knockout of Nox2 had no effect on tumour-vessel density. As underlying mechanisms, we identified a defect in hypoxia signalling in Nox4-/- mice. Hypoxia-inducible factor 1-alpha (Hif-1α) accumulation in the tumours was attenuated as was the expression of the Hif-1α-dependent pro-angiogenic genes vascular endothelial growth factor-A, glucose transporter 1 and adrenomedullin.
CONCLUSION
By regulating the tumour-vessel density through stabilization of Hif-1α and induction of VEGF expression, Nox4 promotes tumour angiogenesis and may represent a novel target for anti-angiogenic tumour therapy.
Topics: Animals; Blotting, Western; Disease Models, Animal; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Oxidase 4; NADPH Oxidases; Neoplasms; Neovascularization, Pathologic; Polymerase Chain Reaction
PubMed: 26513738
DOI: 10.1111/apha.12625 -
Se Pu = Chinese Journal of... Oct 2022A new method for sample pretreatment using improved QuEChERs was established, and 289 organic pollutants with health risks could be identified and quantified through gas...
A new method for sample pretreatment using improved QuEChERs was established, and 289 organic pollutants with health risks could be identified and quantified through gas chromatography-orbitrap high-resolution mass spectrometry (GC-Orbitrap HRMS). A high-resolution database of 289 environmental pollutants belonging to ten categories, including organochlorine pesticides (OCPs), polycyclic aromatic hydrocarbons (PAHs), phthalates (PAEs), polychlorinated biphenyls (PCBs), and other agricultural chemicals (ACs), was established for the non-targeted screening and quantitative analysis. A simple method for biological sample preparation using improved QuEChERs was proposed by combining a conventional QuEChERs method and a column purification method. After purification using a Florisil column, the lipid content was reduced by 99.9%, which significantly reduced the interference of the matrix effect observed during the analysis. Furthermore, simultaneous high-accuracy qualitative screening and quantitative analysis of the target compounds were performed through high-resolution mass spectrometry (60000 resolution) conducted in the full scan mode. The limits of quantification were 0.56-57.8 pg/g, presenting a large linear range (~10), and the recovery range was 40%-120%. Due to the high-resolution and sensitivity of Q Exactive GC-Orbitrap HRMS, the limits of quantification of the target compounds were significantly lower than those achieved through methods based on conventional chromatography and mass spectrometry. Moreover, ultratrace organic contaminants that cannot be detected by conventional methods can be accurately quantified by the proposed method. Sea cucumber samples collected at the breeding site were analyzed using the proposed high-coverage multi-objective analytical method, and more than 100 types of organic pollutants were detected; the mean contents of PAHs, ACs, PAEs, and OCPs were 157.8, 153.2, 64.4, and 46.4 ng/g dw, respectively, which were higher than those of other pollutants. Some new contaminants, such as 9-chlorofluorene, 5-chloroacenaphthene, and 3-methylcholanthrene, were detected at very low contents for the first time in the sea cucumber samples. The proposed method is simple and efficient, allows the detection of pollutants at very low contents, and provides accurate and reliable results. Thus, this high-coverage multi-objective analytical method can be widely used for broad-spectrum screening and accurate quantification of contaminants in various aquatic products, providing technical support for food safety control.
Topics: Animals; Environmental Pollutants; Gas Chromatography-Mass Spectrometry; Hydrocarbons, Chlorinated; Lipids; Mass Spectrometry; Methylcholanthrene; Pesticides; Polychlorinated Biphenyls; Polycyclic Aromatic Hydrocarbons; Sea Cucumbers
PubMed: 36222258
DOI: 10.3724/SP.J.1123.2022.04001 -
Proceedings of the National Academy of... Jan 2018Exposure of certain cell lines to methylcholanthrene, X-rays, or physiological growth constraint leads to preneoplastic transformation in all or most of the treated... (Review)
Review
Exposure of certain cell lines to methylcholanthrene, X-rays, or physiological growth constraint leads to preneoplastic transformation in all or most of the treated cells. After attaining confluence, a fraction in those cells progress to full transformation, as evidenced by their ability to form discrete foci distinguishable from the surrounding cells by virtue of their higher density. Transformation induced by suspension in agar, an even stronger growth-selective condition than confluence, is reminiscent of all but the final differentiated stage of a normal developmental process, epithelial-mesenchymal transition. Changes associated with transformation are not restricted to focus-forming cells, as the permissiveness for focus formation provided by confluent cells surrounding transformed foci is greater than that of nonselected cells. The neoplastic process can also be reversed in culture. Transformed cells passaged at low density in high serum revert to normal morphology and growth behavior in vitro and lose the capacity for tumor formation in vivo. We propose that transformation and its reversal are driven by a process of phenotypic selection that involves entire heterogeneous populations of cells responding to microenvironmental changes. Because of the involvement of whole cell populations, we view this process as fundamentally adaptive and epigenetic in nature.
Topics: Adaptation, Biological; Animals; Cell Transformation, Neoplastic; Cellular Microenvironment; Epigenesis, Genetic; Phenotype; Selection, Genetic
PubMed: 29311337
DOI: 10.1073/pnas.1717299115 -
International Journal of Molecular... Oct 2017Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and its expression is influenced by environmental compounds, such as 3-methylcholanthrene...
Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and its expression is influenced by environmental compounds, such as 3-methylcholanthrene (3-MC) and β-naphthoflavone (β-NF). AhR and its downstream genes, such as , are considered to play a pivotal role in xenobiotic responses. AhR signaling has also been proposed to mediate osteogenesis in experimental animals, but its details have remained unclear. Therefore, in this study, we examined the possible roles of AhR in human bone. Immunohistochemical analysis revealed that AhR was detected in both osteoblasts and osteoclasts. We then screened AhR-target genes using a microarray analysis in human osteoblastic hFOB cells. Results of microarray and subsequent PCR analysis did reveal that estrogen metabolizing and synthesizing enzymes, such as CYP1B1 and aromatase, were increased by 3-MC in hFOB and osteosarcoma cell line, MG-63. The subsequent antibody cytokine analysis also demonstrated that interleukin-1β and -6 expression was increased by 3-MC and β-NF in hFOB cells and these interleukins were well known to induce aromatase. We then examined the cell proliferation rate of hFOB and MG-63 cells co-treated with 3-MC and testosterone as an aromatase substrate. The status of cell proliferation in both hFOB and MG-63 cells was stimulated by 3-MC and testosterone treatment, which was also inhibited by an estrogen blocker, aromatase inhibitor, or AhR antagonist. These findings indicated that AhR could regulate estrogen synthesis and metabolism in bone tissues through cytokine/aromatase signaling.
Topics: Aromatase; Bone and Bones; Cell Line; Cell Line, Tumor; Cell Proliferation; Cytokines; Estrogens; Gene Expression; Gene Expression Regulation; Humans; Immunohistochemistry; Methylcholanthrene; Osteoblasts; Osteosarcoma; Receptors, Aryl Hydrocarbon; Signal Transduction
PubMed: 29039776
DOI: 10.3390/ijms18102159 -
American Journal of Cancer Research 2023The expression status of mineralocorticoid receptor (MR) and its biological significance in human urothelial carcinoma remain unknown. The present study aimed to...
The expression status of mineralocorticoid receptor (MR) and its biological significance in human urothelial carcinoma remain unknown. The present study aimed to determine the functional role of MR in the development of urothelial cancer. In human normal urothelial SVHUC cells with exposure to a chemical carcinogen 3-methylcholanthrene (MCA), we assessed the effects of a natural MR ligand, aldosterone, and 3 MR antagonists, including spironolactone, eplerenone, and esaxerenone, as well as knockdown of MR via shRNA virus infection, on their neoplastic/malignant transformation. The system with carcinogen challenge showed that aldosterone and anti-mineralocorticoids significantly prevented and promoted, respectively, the neoplastic transformation of SVHUC cells. Similarly, MR knockdown in SVHUC cells considerably induced MCA-mediated neoplastic transformation, compared with a control subline. In addition, MR knockdown or antagonist treatment resulted in increases in the expression of β-catenin, c-Fos, and N-cadherin, and a decrease in that of E-cadherin. Meanwhile, spironolactone, which is known to possess anti-androgenic activity, rather suppressed the neoplastic transformation of a SVHUC subline stably expressing wild-type androgen receptor, indicating its dominant effect via the androgen receptor pathway. Immunohistochemistry in surgical specimens detected MR signals in 77 (98.7%; 23.1% weak/1+, 42.3% moderate/2+, and 33.3% strong/3+) of 78 non-invasive bladder tumors, which was significantly (<0.001) lower than in adjacent non-neoplastic urothelial tissues (100%; 20.5% 2+ and 79.5% 3+). Moreover, the risks for disease recurrence after transurethral surgery were marginally lower in female patients with MR-high (2+/3+) tumor (=0.068) and significantly lower in all patients with MR-high/glucocorticoid receptor-high tumor (=0.025), compared with respective controls. These findings suggest that MR signaling functions as a suppressor for urothelial tumorigenesis.
PubMed: 36895984
DOI: No ID Found -
Scientific Reports May 2016In vitro liver models have been important tools for more than 40 years for academic research and preclinical toxicity screening by the pharmaceutical industry....
In vitro liver models have been important tools for more than 40 years for academic research and preclinical toxicity screening by the pharmaceutical industry. Hepatocytes, the highly metabolic parenchymal cells of the liver, are efficient at different metabolic chemistries depending on their relative spatial location along the sinusoid from the portal triad to the central vein. Although replicating hepatocyte metabolic zonation is vitally important for physiologically-relevant in vitro liver tissue and organ models, it is most often completely overlooked. Here, we demonstrate the creation of spatially-controlled zonation across multiple hepatocyte metabolism levels through the application of precise concentration gradients of exogenous hormone (insulin and glucagon) and chemical (3-methylcholanthrene) induction agents in a microfluidic device. Observed gradients in glycogen storage via periodic acid-Schiff staining, urea production via carbamoyl phosphatase synthetase I staining, and cell viability after exposure to allyl alcohol and acetaminophen demonstrated the in vitro creation of hepatocyte carbohydrate, nitrogen, alcohol degradation, and drug conjugation metabolic zonation. This type of advanced control system will be crucial for studies evaluating drug metabolism and toxicology using in vitro constructs.
Topics: Acetaminophen; Animals; Cell Culture Techniques; Cells, Cultured; Ethanol; Female; Glucagon; Glucose; Hepatocytes; Insulin; Lab-On-A-Chip Devices; Methylcholanthrene; Microtechnology; Nitrogen; Rats, Inbred Lew
PubMed: 27240736
DOI: 10.1038/srep26868 -
Journal of Experimental & Clinical... Aug 2019The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1,...
BACKGROUND
The 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.
METHODS
Molecular 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.
RESULTS
We 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.
CONCLUSIONS
In 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.
Topics: Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Proliferation; Cytochrome P-450 CYP1B1; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Methylcholanthrene; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; Protein Transport; Receptors, Aryl Hydrocarbon; Receptors, Estrogen; Receptors, G-Protein-Coupled; Signal Transduction; Structure-Activity Relationship
PubMed: 31370872
DOI: 10.1186/s13046-019-1337-2 -
International Journal of Cancer Jan 2016Intratumor heterogeneity among cancer cells is promoted by reversible or irreversible genetic alterations and by different microenvironmental factors. There is... (Review)
Review
Intratumor heterogeneity among cancer cells is promoted by reversible or irreversible genetic alterations and by different microenvironmental factors. There is considerable experimental evidence of the presence of a variety of malignant cell clones with a wide diversity of major histocompatibility class I (MHC-I) expression during early stages of tumor development. This variety of MHC-I phenotypes may define the evolution of a particular tumor. Loss of MHC-I molecules frequently results in immune escape of MHC-negative or -deficient tumor cells from the host T cell-mediated immune response. We review here the results obtained by our group and other researchers in animal models and humans, showing how MHC-I intratumor heterogeneity may affect local oncogenicity and metastatic progression. In particular, we summarize the data obtained in an experimental mouse cancer model of a methylcholanthrene-induced fibrosarcoma (GR9), in which isolated clones with different MHC-I expression patterns demonstrated distinct local tumor growth rates and metastatic capacities. The observed "explosion of diversity" of MHC-I phenotypes in primary tumor clones and the molecular mechanism ("hard"/irreversible or "soft"/reversible) responsible for a given MHC-I alteration might determine not only the metastatic capacity of the cells but also their response to immunotherapy. We also illustrate the generation of further MHC heterogeneity during metastatic colonization and discuss different strategies to favor tumor rejection by counteracting MHC-I loss. Finally, we highlight the role of MHC-I genes in tumor dormancy and cell cycle control.
Topics: Animals; Histocompatibility Antigens Class I; Humans; Neoplasms; Phenotype; Tumor Escape
PubMed: 25471439
DOI: 10.1002/ijc.29375