Dysregulation of gap junction intercellular communication (GJIC) is recognized as one of the key hallmarks for identifying non-genotoxic carcinogens (NGTxC). Currently, there is a demand for in vitro assays addressing the gap junction hallmark, which would have the potential to eventually become an integral part of an integrated approach to the testing and assessment (IATA) of NGTxC. The scrape loading-dye transfer (SL-DT) technique is a simple assay for the functional evaluation of GJIC in various in vitro cultured mammalian cells and represents an interesting candidate assay. Out of the various techniques for evaluating GJIC, the SL-DT assay has been used frequently to assess the effects of various chemicals on GJIC in toxicological and tumor promotion research. In this review, we systematically searched the existing literature to gather papers assessing GJIC using the SL-DT assay in a rat liver epithelial cell line, WB-F344, after treating with chemicals, especially environmental and food toxicants, drugs, reproductive-, cardio- and neuro-toxicants and chemical tumor promoters. We discuss findings derived from the SL-DT assay with the known knowledge about the tumor-promoting activity and carcinogenicity of the assessed chemicals to evaluate the predictive capacity of the SL-DT assay in terms of its sensitivity, specificity and accuracy for identifying carcinogens. These data represent important information with respect to the applicability of the SL-DT assay for the testing of NGTxC within the IATA framework.

• Klíčová slova
• carcinogenesis, carcinogens, gap junction intercellular communication, scrape loading-dye transfer,
• MeSH
• barvicí látky metabolismus   MeSH
• biotest metody   MeSH
• buněčné linie MeSH
• fluorescenční mikroskopie metody   MeSH
• játra patologie   MeSH
• karcinogeny MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• mezerový spoj metabolismus   MeSH
• mezibuněčná komunikace účinky léků   fyziologie   MeSH
• testy karcinogenity metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• barvicí látky MeSH
• karcinogeny MeSH

Inhalation exposures to polycyclic aromatic hydrocarbons (PAHs) have been associated with various adverse health effects, including chronic lung diseases and cancer. Using human bronchial epithelial cell line HBE1, we investigated the effects of structurally different PAHs on tissue homeostatic processes, namely gap junctional intercellular communication (GJIC) and MAPKs activity. Rapid (<1 h) and sustained (up to 24 h) inhibition of GJIC was induced by low/middle molecular weight (MW) PAHs, particularly by those with a bay- or bay-like region (1- and 9-methylanthracene, fluoranthene), but also by fluorene and pyrene. In contrast, linear low MW (anthracene, 2-methylanthracene) or higher MW (chrysene) PAHs did not affect GJIC. Fluoranthene, 1- and 9-methylanthracene induced strong and sustained activation of MAPK ERK1/2, whereas MAPK p38 was activated rather nonspecifically by all tested PAHs. Low/middle MW PAHs can disrupt tissue homeostasis in human airway epithelium via structure-dependent nongenotoxic mechanisms, which can contribute to their human health hazards.

• Klíčová slova
• Gap junctional intercellular communication, Human bronchial epithelial cell line, Methylated anthracenes, Mitogen-activated protein kinases, Nongenotoxic mechanisms, Polycyclic aromatic hydrocarbons,
• MeSH
• bronchy cytologie   MeSH
• buněčné linie MeSH
• epitelové buňky účinky léků   fyziologie   MeSH
• lidé MeSH
• mezerový spoj účinky léků   MeSH
• mezibuněčná komunikace účinky léků   MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• polycyklické aromatické uhlovodíky toxicita   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mitogenem aktivované proteinkinasy MeSH
• polycyklické aromatické uhlovodíky MeSH

Humans are exposed to phthalates released from plastics, cosmetics, or food on a daily basis. Phthalates have low acute liver toxicity, but their chronic exposures could induce molecular and cellular effects linked to adverse health outcomes, such as liver tumor promotion or chronic liver diseases. The alternation of gap junctional intercellular communication (GJIC) and MAPK-Erk1/2 pathways in liver progenitor or oval cells can disrupt liver tissue homeostatic mechanisms and affect the development and severity of these adverse outcomes. Our study with 20 different phthalates revealed their structurally dependent effects on liver GJIC and MAPK-Erk1/2 signaling in rat liver WB-F344 cell line with characteristics of liver oval cells. The phthalates with a medium-length side chain (3-6 C) were the most potent dysregulators of GJIC and activators of MAPK-Erk1/2. The effects occurred rapidly, suggesting the activation of non-genomic (non-transcriptional) mechanisms directly by the parental compounds. Short-chain phthalates (1-2 C) did not dysregulate GJIC even after longer exposures and did not activate MAPK-Erk1/2. Longer chain (≥7 C) phthalates, such as DEHP or DINP, moderately activated MAPK-Erk1/2, but inhibited GJIC only after prolonged exposures (>12 h), suggesting that GJIC dysregulation occurs via genomic mechanisms, or (bio)transformation. Overall, medium-chain phthalates rapidly affected the key tissue homeostatic mechanisms in the liver oval cell population via non-genomic pathways, which might contribute to the development of chronic liver toxicity and diseases.

• Klíčová slova
• MAP-kinases Erk1/2 activation, gap junctional intercellular communication, gap junctions, hepatotoxicity, non-genomic mechanism, oval cells, phthalates, progenitor cells,
• MeSH
• buněčné linie MeSH
• játra cytologie   účinky léků   metabolismus   MeSH
• krysa rodu Rattus MeSH
• kyseliny ftalové aplikace a dávkování   chemie   toxicita   MeSH
• MAP kinasový signální systém účinky léků   MeSH
• mezerový spoj účinky léků   MeSH
• mezibuněčná komunikace účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyseliny ftalové MeSH

Gap junctional intercellular communication (GJIC) is a vital cellular process required for maintenance of tissue homeostasis. In vitro assessment of GJIC represents valuable phenotypic endpoint that could be effectively utilized as an integral component in modern toxicity testing, drug screening or biomedical in vitro research. However, currently available methods for quantifying GJIC with higher-throughputs typically require specialized equipment, proprietary software and/or genetically engineered cell models. To overcome these limitations, we present here an innovative adaptation of traditional, fluorescence microscopy-based scrape loading-dye transfer (SL-DT) assay, which has been optimized to simultaneously evaluate GJIC, cell density and viability. This multiparametric method was demonstrated to be suitable for various multiwell microplate formats, which facilitates an automatized image acquisition. The assay workflow is further assisted by an open source-based software tools for batch image processing, analysis and evaluation of GJIC, cell density and viability. Our results suggest that this approach provides a simple, fast, versatile and cost effective way for in vitro high-throughput assessment of GJIC and other related phenotypic cellular events, which could be included into in vitro screening and assessment of pharmacologically and toxicologically relevant compounds.

• MeSH
• fluorescenční mikroskopie metody   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• mezerový spoj * MeSH
• mezibuněčná komunikace * MeSH
• molekulární zobrazování metody   MeSH
• počet buněk * MeSH
• počítačové zpracování obrazu metody   MeSH
• viabilita buněk * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Klíčová slova
• endocrine disruptors, epigenetic toxicity, gap junctional intercellular communication, mitogen-activated protein kinases, non-genomic signaling., phosphatidylcholine-specific phospholipase C,
• 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
• Názvy látek
• androgenní receptory MeSH
• insekticidy MeSH
• konexin 43 MeSH
• methoxychlor MeSH
• mitogenem aktivované proteinkinasy p38 MeSH
• oxazoly MeSH
• receptory pro estrogeny MeSH
• vinclozolin MeSH Prohlížeč

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
• Názvy látek
• antikarcinogenní látky MeSH
• DDT MeSH
• fluoranthene MeSH Prohlížeč
• fluoreny MeSH
• fluorokarbony MeSH
• hexachlorcyklohexan MeSH
• kapryláty MeSH
• karcinogeny MeSH
• kurkumin MeSH
• metformin MeSH
• perfluorooctanoic acid MeSH Prohlížeč
• tetradekanoylforbolacetát 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
• norbornany MeSH
• potkani inbrední F344 MeSH
• přemostěné cyklické sloučeniny farmakologie   MeSH
• resveratrol MeSH
• stilbeny farmakologie   MeSH
• thiokarbamáty 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
• Názvy látek
• butadieny MeSH
• fosfatidylcholiny MeSH
• fosfolipasy typu C MeSH
• nitrily MeSH
• norbornany MeSH
• phosphatidylcholine-specific phospholipase C MeSH Prohlížeč
• přemostěné cyklické sloučeniny MeSH
• resveratrol MeSH
• stilbeny MeSH
• thiokarbamáty MeSH
• thioketony MeSH
• tricyclodecane-9-yl-xanthogenate MeSH Prohlížeč
• U 0126 MeSH Prohlížeč

Toxicity and liver tumor promotion of cyanotoxins microcystins have been extensively studied. However, recent studies document that other metabolites present in the complex cyanobacterial water blooms may also have adverse health effects. In this study we used rat liver epithelial stem-like cells (WB-F344) to examine the effects of cyanobacterial extracts on two established markers of tumor promotion, inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) - ERK1/2. Extracts of cyanobacteria (laboratory cultures of Microcystis aeruginosa and Aphanizomenon flos-aquae and water blooms dominated by these species) inhibited GJIC and activated MAPKs in a dose-dependent manner (effective concentrations ranging 0.5-5mgd.w./mL). Effects were independent of the microcystin content and the strongest responses were elicited by the extracts of Aphanizomenon sp. Neither pure microcystin-LR nor cylindrospermopsin inhibited GJIC or activated MAPKs. Modulations of GJIC and MAPKs appeared to be specific to cyanobacterial extracts since extracts from green alga Chlamydomonas reinhardtii, heterotrophic bacterium Klebsiella terrigena, and isolated bacterial lipopolysaccharides had no comparable effects. Our study provides the first evidence on the existence of unknown cyanobacterial toxic metabolites that affect in vitro biomarkers of tumor promotion, i.e. inhibition of GJIC and activation of MAPKs.

• MeSH
• aktivace enzymů účinky léků   MeSH
• alkaloidy MeSH
• Aphanizomenon chemie   izolace a purifikace   MeSH
• bakteriální toxiny MeSH
• buněčné linie MeSH
• časové faktory MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fosforylace účinky léků   MeSH
• karcinogeny chemie   toxicita   MeSH
• komplexní směsi chemie   toxicita   MeSH
• krysa rodu Rattus MeSH
• mezerový spoj účinky léků   MeSH
• mezibuněčná komunikace účinky léků   MeSH
• Microcystis chemie   izolace a purifikace   MeSH
• mikrocystiny analýza   toxicita   MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• sinice chemie   izolace a purifikace   MeSH
• sladká voda mikrobiologie   MeSH
• toxiny kmene Cyanobacteria MeSH
• uracil analogy a deriváty   toxicita   MeSH
• vztah mezi dávkou a účinkem léčiva 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
• srovnávací studie MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• alkaloidy MeSH
• bakteriální toxiny MeSH
• cylindrospermopsin MeSH Prohlížeč
• extracelulárním signálem regulované MAP kinasy MeSH
• karcinogeny MeSH
• komplexní směsi MeSH
• mikrocystiny MeSH
• mitogenem aktivované proteinkinasy MeSH
• toxiny kmene Cyanobacteria MeSH
• uracil MeSH