stilbenes and alpha-naphthoflavone

Infertility is a global health problem with an estimated incidence of 15%. Exposure to chemicals is a potential causal factor, and there is a lack of studies examining the effects on female germ cells. Here, we have studied the impact of different aryl hydrocarbon receptor (AHR) modulators on human ovarian follicles using a human ovarian tissue culture model. Expression of AHR was analyzed in tissue samples, and effects of the selected ligands resveratrol (RSVL), 6-formylindolo(3,2-b)carbazole (FICZ), and alpha-naphthoflavone (aNF) on AHR transactivation studied in a granulosa cell tumor line. Cortical human ovarian tissue containing preantral follicles was exposed to the ligands or vehicle (dimethylsulfoxide, DMSO) for seven days in vitro. Follicle growth was assessed by counting and measuring follicles from serial tissue sections, cell death quantified using in situ Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay, and steroid hormone production measured using a newly developed ultra-performance liquid chromatography method. AHR was expressed in all donated ovarian tissue samples. FICZ induced AHR transactivation in the granulosa cell line while aNF antagonised it. Compared to DMSO control, FICZ had no effect on follicles in culture, RSVL increased the proportion of growing follicles, and aNF increased cell death, disrupted growth of secondary follicles, increased testosterone, and reduced estradiol levels. We conclude that RSVL supports and aNF disrupts growth of human ovarian follicles in culture. We further conclude that the human ovarian tissue culture model is suitable for studying effects of chemicals on follicular biology.

Topics: Adult; Benzoflavones; Carbazoles; Cell Death; Female; Humans; In Situ Nick-End Labeling; Ovarian Follicle; Receptors, Aryl Hydrocarbon; Resveratrol; Stilbenes; Tissue Culture Techniques

Human papilloma viruses 16 and 18 express E6 and E7 oncoproteins. E6 activates and redirects E6-associated protein (E6AP), an E3 ubiquitin ligase. E6AP interacts with Ube2l3, an E2 ubiquitin conjugating enzyme protein (also known as UbcH7), to promote p53 ubiquitination and degradation by the 26S proteasome. Therefore, blocking E6-mediated p53 degradation might be an alternative treatment for cervical cancer. In addition, activation of the aryl hydrocarbon receptor (AHR) induces Ube2l3 expression, resulting in p53 ubiquitination and degradation. The aim of the present study was to determine whether inhibition of AHR in HeLa cells resulted in an increase in p53 and apoptosis along with a decrease in cell proliferation. The results demonstrate that two AHR antagonists, α-naphthoflavone (α-NF) and resveratrol, decreased cell proliferation, arrested cells in the gap 1/synthesis (G1/S) phases, and increased p53 levels and apoptosis. However, knocking out the Ahr gene did not abrogate the effects of α-NF and resveratrol. Moreover, Ahr-null cells presented similar cell proliferation rates and apoptosis levels when compared to control HeLa cells. Taken together, the results indicate that α-NF's and resveratrol's cytostatic and cytotoxic actions, respectively, occur through an AHR-independent mechanism, and that AHR is not required for HeLa cell proliferation.

Topics: Apoptosis; Benzoflavones; Cell Proliferation; CRISPR-Cas Systems; G1 Phase Cell Cycle Checkpoints; HeLa Cells; Humans; Microscopy, Confocal; Receptors, Aryl Hydrocarbon; Resveratrol; Stilbenes; Tumor Suppressor Protein p53

Developmental exposure to aryl hydrocarbon receptor (AhR) agonists in fish causes severe defects in the cardiovascular system. However, the effects of acute AhR agonist exposure on the adult fish cardiovascular system are not clear. We hypothesized that AhR-mediated changes in adult vascular tissue gene expression would differ from that of hepatic tissue. Therefore, zebrafish (Danio rerio) were intraperitoneally injected with the AhR agonists benzo-a-pyrene (BaP; 1mg/kg) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 20microg/kg) alone and in combination with the AhR antagonists resveratrol (Res; 10mg/kg) or alpha-naphthoflavone (ANF; 50mg/kg). Hepatic and mesenteric artery cytochrome P450 enzyme (subtypes 1A, 1B1, 1C1, and 1C2) and cyclooxygenase enzyme (subtypes 1, 2a, and 2b) mRNA expression was quantified using real-time reverse transcriptase PCR. TCDD exposure significantly increased (p

Topics: Animals; Benzo(a)pyrene; Benzoflavones; Cytochrome P-450 Enzyme System; Gene Expression Regulation; Liver; Mesenteric Arteries; Polychlorinated Dibenzodioxins; Prostaglandin-Endoperoxide Synthases; Receptors, Aryl Hydrocarbon; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stilbenes; Zebrafish

The arylhydrocarbon receptor (AhR) is known to mediate toxic responses to dioxin (2,3,7,8-tetrachlorodibenzo-p- dioxin) and related compounds and has been extensively characterized from a toxicological viewpoint. However, it has recently been reported that the AhR may have a central role in ovarian physiology. To investigate the role of AhR during oocyte maturation, we analyzed the expression of AhR, its nuclear partner AhR nuclear translocator, and the major target gene CYP1A1, in bovine cumulus-oocyte complexes (COCs) by semiquantitative RT-PCR and Western blot. Coexpression of AhR and AhR nuclear translocator was observed in both oocytes and surrounding cumulus cells before and after in vitro maturation (IVM). Furthermore, after IVM, both cell types showed a clear up-regulation of AhR mRNA compared with the expression at 0 h. Constitutive expression of CYP1A1 mRNA was observed in immature oocytes at the background level, whereas no expression was observed in the surrounding cumulus cells. Interestingly, a significant increase in CYP1A1 expression level was observed in both oocytes and cumulus cells after IVM. To further investigate the role of AhR in CYP1A1 up-regulation and oocyte maturation, COCs were treated throughout IVM with the AhR antagonists, alpha-naphthoflavone and resveratrol. Both antagonists decreased the level of CYP1A1 in COCs compared with controls. Furthermore, CYP1A1 down-regulation was accompanied by a reduced ability of oocytes to complete in vitro maturation until metaphase II stage. These results suggest that CYP1A1 induction in COCs is necessary for correct proceeding of in vitro oocyte maturation in bovine and suggest a physiological role of AhR during resumption of meiosis.

Topics: Animals; Aryl Hydrocarbon Receptor Nuclear Translocator; Benzoflavones; Blood; Cattle; Cellular Senescence; Cytochrome P-450 CYP1A1; DNA-Binding Proteins; Female; Gene Expression; Oocytes; Receptors, Aryl Hydrocarbon; Resveratrol; RNA, Messenger; Signal Transduction; Stilbenes; Transcription Factors

The aryl hydrocarbon receptor (AHR) is a member of ligand-activated transcription factors and conserved among vertebrates. To investigate the role of AHR in fish development, medaka embryos were treated with agonist (2,3,7,8-tetrachlorodibenzo-p-dioxin), antagonists (alpha-naphthoflavone and resveratrol), and inhibitor (piperonyl butoxide) of cytochromes (Cyts) P450 encoded by a battery of target genes. These embryos were found to have similar abnormal phenotypes. Among the most consistent phenotypes were blood clotting and malformation of bone that were associated with vascular damages. These results thus indicate that control of AHR is important for proper development of fish embryos. AHR may control levels of Cyts P450 that are responsible for synthesis and metabolism of a toxic compound that caused the abnormal phenotypes. Complementary DNA fragments encoding AHR homologs were cloned from medaka embryos. AHR-specific mRNA was ubiquitously expressed in embryos and adult tissues.

Topics: Acetylcysteine; Animals; Base Sequence; Benzoflavones; Blood Coagulation; Blood Vessels; Bone and Bones; Conserved Sequence; DNA, Complementary; Evolution, Molecular; Gene Expression Regulation; Oryzias; Phenotype; Piperonyl Butoxide; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Resveratrol; RNA, Messenger; Sequence Homology, Nucleic Acid; Stilbenes

The metabolism of testosterone and androstenedione by liver microsomes was investigated after treatment of rats with trans-stilbene oxide, phenobarbital, or 3-methylcholanthrene. Conditions for linearity of the assay with time and amount of cytochrome P-450, as well as saturating substrate concentrations, were established. The metabolites were separated by thin-layer chromatography and quantitated by scintillation counting. The rates of formation of different testosterone and androstenedione metabolites after induction with trans-stilbene oxide or phenobarbital were similar, indicating that these xenobiotics induce the same isozyme of cytochrome P-450. This conclusion was further supported using inhibitors of cytochrome P-450 (SKF-525A, metyrapone and alpha-naphthoflavone) and with immunoinhibition by antibodies directed towards the phenobarbital-inducible form of cytochrome P-450. After treatment with trans-stilbene oxide or phenobarbital, the specific rates of formation of the 6 beta- and/or 2 beta-hydroxy metabolites and of 17 beta-hydroxy-4-androstene-3,16-dione were increased. In contrast, administration of 3-methylcholanthrene led to decreases in the specific rates of formation of almost all testosterone and androstenedione metabolites investigated. However, all three of these inducers cause increases in the total liver metabolism of testosterone and androstenedione. These increases are 2--30-fold in the case of trans-stilbene oxide, 3--46-fold for phenobarbital and 1--4-fold after treatment with 3-methylcholanthrene. The possible physiological significance of these effects is as yet unknown.

Topics: Androstenedione; Animals; Benzoflavones; Cytochrome P-450 Enzyme System; Hydroxylation; Kinetics; Male; Methylcholanthrene; Metyrapone; Microsomes, Liver; Phenobarbital; Proadifen; Rats; Rats, Inbred Strains; Stilbenes; Testosterone