Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 µM for MXC and 126 µM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER- or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors.
- MeSH
- androgenní receptory metabolismus MeSH
- buněčné linie MeSH
- insekticidy toxicita MeSH
- játra cytologie účinky léků metabolismus MeSH
- kmenové buňky účinky léků metabolismus MeSH
- konexin 43 metabolismus MeSH
- krysa rodu rattus MeSH
- MAP kinasový signální systém účinky léků MeSH
- methoxychlor toxicita MeSH
- mezerový spoj účinky léků MeSH
- mezibuněčná komunikace účinky léků MeSH
- mitogenem aktivované proteinkinasy p38 metabolismus MeSH
- oxazoly toxicita MeSH
- potkani inbrední F344 MeSH
- receptory pro estrogeny metabolismus MeSH
- signální transdukce účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Altered gap junctional intercellular communication (GJIC) has been associated with chemical carcinogenesis, where both chemical tumor promoters and chemopreventive agents (CPAs) are known to conversely modulate GJIC. The aim of this study was to investigate whether attenuation of chemically inhibited GJIC represents a common outcome induced by different CPAs, which could be effectively evaluated using in vitro methods. Rat liver epithelial cells WB-F344 were pretreated with a CPA for either 30 min or 24 h, and then exposed to GJIC-inhibiting concentration of a selected tumor promoter or environmental toxicant [12-O-tetradecanoylphorbol-13-acetate (TPA), lindane, fluoranthene, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), perfluorooctanoic acid (PFOA), or pentachlorophenol]. Out of nine CPAs tested, quercetin and silibinin elicited the most pronounced effects, preventing the dysregulation of GJIC by all the GJIC inhibitors, but DDT. Metformin and curcumin attenuated the effects of three GJIC inhibitors, whereas the other CPAs prevented the effects of two (diallyl sulfide, emodin) or one (indole-3-carbinol, thymoquinone) GJIC inhibitor. Significant attenuation of chemically induced inhibition of GJIC was observed in 27 (50%) out of 54 possible combinations of nine CPAs and six GJIC inhibitors. Our data demonstrate that in vitro evaluation of GJIC can be used as an effective screening tool for identification of chemicals with potential chemopreventive activity.
- MeSH
- antikarcinogenní látky farmakologie MeSH
- DDT toxicita MeSH
- epitelové buňky účinky léků MeSH
- fluoreny toxicita MeSH
- fluorokarbony toxicita MeSH
- hexachlorcyklohexan toxicita MeSH
- játra cytologie účinky léků MeSH
- kapryláty toxicita MeSH
- karcinogeny toxicita MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- kurkumin farmakologie MeSH
- metformin farmakologie MeSH
- mezerový spoj účinky léků metabolismus MeSH
- mezibuněčná komunikace účinky léků MeSH
- potkani inbrední F344 MeSH
- tetradekanoylforbolacetát toxicita MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The scrape loading/dye transfer (SL/DT) technique is a simple functional assay for the simultaneous assessment of gap junctional intercellular communication (GJIC) in a large population of cells. The equipment needs are minimal and are typically met in standard cell biology labs, and SL/DT is the simplest and quickest of all the assays that measure GJIC. This assay has also been adapted for in vivo studies. The SL/DT assay is also conducive to a high-throughput setup with automated fluorescence microscopy imaging and analysis to elucidate more samples in shorter time, and hence can serve a broad range of in vitro pharmacological and toxicological needs.
- MeSH
- dextrany metabolismus MeSH
- fluorescenční barviva metabolismus MeSH
- fluorescenční mikroskopie metody MeSH
- isochinoliny metabolismus MeSH
- kultivované buňky MeSH
- mezerový spoj metabolismus ultrastruktura MeSH
- mezibuněčná komunikace * MeSH
- myši MeSH
- rhodaminy metabolismus MeSH
- Sertoliho buňky MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
UNLABELLED: Dysregulation of gap junctional intercellular communication (GJIC) has been associated with different pathologies, including cancer; however, molecular mechanisms regulating GJIC are not fully understood. Mitogen Activated Protein Kinase (MAPK)-dependent mechanisms of GJIC-dysregulation have been well-established, however recent discoveries have implicated phosphatidylcholine-specific phospholipase C (PC-PLC) in the regulation of GJIC. What is not known is how prevalent these two signaling mechanisms are in toxicant/toxin-induced dysregulation of GJIC, and do toxicants/toxins work through either signaling mechanisms or both, or through alternative signaling mechanisms. Different chemical toxicants were used to assess whether they dysregulate GJIC via MEK or PC-PLC, or both Mek and PC-PLC, or through other signaling pathways, using a pluripotent rat liver epithelial oval-cell line, WB-F344. Epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, thrombin receptor activating peptide-6 and lindane regulated GJIC through a MEK1/2-dependent mechanism that was independent of PC-PLC; whereas PAHs, DDT, PCB 153, dicumylperoxide and perfluorodecanoic acid inhibited GJIC through PC-PLC independent of Mek. Dysregulation of GJIC by perfluorooctanoic acid and R59022 required both MEK1/2 and PC-PLC; while benzoylperoxide, arachidonic acid, 18β-glycyrrhetinic acid, perfluorooctane sulfonic acid, 1-monolaurin, pentachlorophenol and alachlor required neither MEK1/2 nor PC-PLC. Resveratrol prevented dysregulation of GJIC by toxicants that acted either through MEK1/2 or PC-PLC. Except for alachlor, resveratrol did not prevent dysregulation of GJIC by toxicants that worked through PC-PLC-independent and MEK1/2-independent pathways, which indicated at least two other, yet unidentified, pathways that are involved in the regulation of GJIC. IN CONCLUSION: the dysregulation of GJIC is a contributing factor to the cancer process; however the underlying mechanisms by which gap junction channels are closed by toxicants vary. Thus, accurate assessments of risk posed by toxic agents, and the role of dietary phytochemicals play in preventing or reversing the effects of these agents must take into account the specific mechanisms involved in the cancer process.
- MeSH
- analýza hlavních komponent MeSH
- buněčné linie MeSH
- butadieny farmakologie MeSH
- fosfatidylcholiny metabolismus MeSH
- fosfolipasy typu C metabolismus MeSH
- krysa rodu rattus MeSH
- mezerový spoj účinky léků metabolismus MeSH
- nitrily farmakologie MeSH
- potkani inbrední F344 MeSH
- přemostěné cyklické sloučeniny farmakologie MeSH
- stilbeny farmakologie MeSH
- thioketony farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Publikační typ
- abstrakt z konference MeSH
- MeSH
- finanční podpora výzkumu jako téma MeSH
- Publikační typ
- abstrakty MeSH