3-dimensional (3D) cell cultures are being increasingly recognized as physiologically more relevant in vitro models than traditional monolayer cultures, because they better mimic in vivo-like microenvironment, cell-cell and cell-extracellular matrix interactions. Nevertheless, the broader use of 3D models might be limited by requirements for special consumables, equipment, or skills for 3D cell cultures, and by their limited throughput and scalability. In this study, we optimized and adapted a commercially available agarose-micromolding technique to produce scaffold-free spheroid cultures. Brightfield microscopy was used for routine nondestructive and noninvasive evaluation of spheroid formation and growth. The workflow is compatible with manual, as well as high speed automated microscopic image acquisition, and it is supplemented with an in-house developed macro 'Spheroid_Finder' for open source software Fiji to facilitate rapid automated image analysis. This protocol was used to characterize and quantify spheroid formation and growth of two different hepatic cell lines, hTERT immortalized, but non-cancerous, adult human liver stem cell line HL1-hT1, and human hepatocellular carcinoma cell line HepG2, as well as their responses to a model antiproliferative and cytotoxic agent, 5-fluorouracil. The complete protocol provides a simple and ready-to-use solution to initiate scaffold-free spheroid cultures in any laboratory with standard equipment for mammalian in vitro cell culture work. Thus, it allows to increase throughput and scale of spheroid culture experiments, which can be greatly utilized in different areas of biomedical, pharmaceutical and toxicological research.

• MeSH
• antimetabolity antitumorózní farmakologie   toxicita   MeSH
• buněčné kultury MeSH
• buněčné sféroidy MeSH
• buňky Hep G2 MeSH
• časové faktory MeSH
• fluorouracil farmakologie   toxicita   MeSH
• hepatocelulární karcinom farmakoterapie   metabolismus   patologie   MeSH
• játra účinky léků   metabolismus   patologie   MeSH
• kmenové buňky účinky léků   metabolismus   patologie   MeSH
• léky antitumorózní - screeningové testy MeSH
• lidé MeSH
• nádory jater farmakoterapie   metabolismus   patologie   MeSH
• proliferace buněk účinky léků   MeSH
• průběh práce MeSH
• rychlé screeningové testy * MeSH
• testy toxicity MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Cyanotoxins microcystin-LR (MC-LR) and cylindrospermopsin (CYN) represent hazardous waterborne contaminants and potent human hepatotoxins. However, in vitro monolayer cultures of hepatic cell lines were found to recapitulate, poorly, major hepatocyte-specific functions and inadequately predict hepatotoxic effects of MC-LR and CYN. We utilized 3-dimensional (3D), scaffold-free spheroid cultures of human telomerase-immortalized adult liver stem cells HL1-hT1 to evaluate hepatotoxic potential of MC-LR and CYN. In monolayer cultures of HL1-hT1 cells, MC-LR did not induce cytotoxic effects (EC50 > 10 micromol/L), while CYN inhibited cell growth and viability (48h-96h EC50 ≈ 5.5-0.6 micromol/L). Growth and viability of small growing spheroids were inhibited by both cyanotoxins (≥0.1 micromol/L) and were associated with blebbing and disintegration at the spheroid surface. Hepatospheroid damage and viability reduction were observed also in large mature spheroids, with viability 96h-EC50 values being 0.04 micromol/L for MC-LR and 0.1 micromol/L for CYN, and No Observed Effect Concentrations <0.01 micromol/L. Spheroid cultures of adult human liver stem cells HL1-hT1 exhibit sensitivity comparable to cultures of primary hepatocytes and provide a simple, practical, and cost-effective tool, which can be effectively used in environmental and toxicological research, including assessment of hepatotoxic potential and effect-based monitoring of various samples contaminated with toxic cyanobacteria.

• MeSH
• bakteriální toxiny MeSH
• játra MeSH
• kmenové buňky MeSH
• lidé MeSH
• mikrocystiny MeSH
• mořské toxiny * MeSH
• sinice * MeSH
• Check Tag
• lidé 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.

• 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

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

Alkylated polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants. In the present study, we determined levels of monomethylated naphthalenes (MeNap), phenanthrenes (MePhe), and anthracenes (MeAnt) in Czech river sediments. The levels of MePhe generally were lower than the concentrations of phenanthrene. In contrast, both MeNap and MeAnt were found at levels higher than their respective parent compounds in the majority of sampling sites. We then investigated their aryl hydrocarbon receptor (AhR)-mediated activity, accumulation of phosphorylated p53 protein, induction of expression of cytochrome P450 1A1 (CYP1A1), inhibition of gap junctional intercellular communication (GJIC), and effects on cell proliferation in rat liver cell models to evaluate the relative importance of these toxicity mechanisms of low-molecular-weight methylated PAHs. Methylated phenanthrene and anthracene compounds were weak inducers of AhR-mediated activity as determined both in a reporter gene assay system and by detection of the endogenous gene (Cyp1a1) induction. 2-Methylphenanthrene was the most potent AhR ligand. Contribution of MeAnt and MePhe to overall AhR-inducing potencies should be taken into account in PAH-contaminated environments. Nevertheless, their effects on AhR were not sufficient to modulate cell proliferation in a normal rat liver progenitor cell model system. These PAHs only had a marginal effect on p53 phosphorylation at high doses of 1-, 3-, and 9-MePhe as well as 1 MeAnt. On the other hand, both 2- and 9-MeAnt as well as all the MePhe under study were efficient inhibitors of GJIC, suggesting that these compounds might act as tumor promoters. In summary, inhibition of GJIC and partial activation of AhR seem to be the most prominent toxic effects of the methylated PAHs in the present study.

• MeSH
• anthraceny toxicita   MeSH
• cytochrom P-450 CYP1A1 genetika   metabolismus   MeSH
• fenantreny toxicita   MeSH
• financování organizované MeSH
• geologické sedimenty chemie   MeSH
• játra cytologie   patologie   MeSH
• karcinogeny toxicita   MeSH
• krysa rodu rattus MeSH
• látky znečišťující životní prostředí toxicita   MeSH
• metylace MeSH
• mezerový spoj metabolismus   účinky léků   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• naftaleny toxicita   MeSH
• proliferace buněk účinky léků   MeSH
• receptory aromatických uhlovodíků metabolismus   MeSH
• regulace genové exprese fyziologie   účinky léků   MeSH
• řeky MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Geografické názvy
• Česká republika MeSH