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Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor
Pavel Babica1, Rimma Zurabian2, Esha R Kumar3
1*Department of Experimental Phycology and Ecotoxicology, Institute of Botany, Brno 60200, Czech Republic; RECETOX, Faculty of Science, Masaryk University, Brno 62500, Czech Republic; Department of Pediatrics and Human Development, and Institute for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824; pavel.babica@centrum.cz.
Abstract:
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.
Insights
Methoxychlor (MXC) and vinclozolin (VIN) rapidly disrupt cell communication (GJIC) and activate MAPKs, independent of hormone receptors. These effects, mediated by PC-PLC, may contribute to transgenerational epigenetic changes.
Area of Science:
- Toxicology
- Cell Biology
- Epigenetics
Background:
- Endocrine disrupting chemicals (EDCs) like methoxychlor (MXC) and vinclozolin (VIN) are known for epigenetic and transgenerational effects.
- Non-endocrine disruption endpoints, such as intercellular communication, are crucial for understanding EDC toxicity.
- Gap junctional intercellular communication (GJIC) is vital for tissue homeostasis and gene regulation.
Purpose of the Study:
- To investigate the effects of MXC and VIN on GJIC and mitogen-activated protein kinases (MAPKs) in rat liver epithelial cells.
- To elucidate the signaling pathways involved in MXC and VIN-induced GJIC dysregulation.
- To determine if these effects are mediated by estrogen (ER) or androgen receptor (AR) signaling.
Main Methods:
- Exposure of WB-F344 rat liver epithelial cells to MXC and VIN at non-cytotoxic doses.
- Assessment of GJIC using scrape-loading/dye transfer assays.
- Analysis of MAPK activation (ERK1/2, p38) and connexin43 phosphorylation/internalization.
- Use of specific inhibitors (MEK1/2, PC-PLC, p38) and receptor modulators (ER, AR) to probe signaling pathways.
Main Results:
- Both MXC and VIN rapidly inhibited GJIC at non-cytotoxic concentrations.
- MXC caused prolonged GJIC inhibition, while VIN's effects were transient.
- VIN induced connexin43 hyperphosphorylation and internalization; both chemicals activated ERK1/2 and p38 MAPKs.
- GJIC inhibition was mediated by phosphatidylcholine-specific phospholipase C (PC-PLC) activation, not MEK1/2, and was independent of ER/AR signaling.
Conclusions:
- MXC and VIN dysregulate GJIC through rapid PC-PLC activation, independent of ER/AR genomic signaling.
- These non-genomic effects on intercellular and intracellular signaling may contribute to the transgenerational epigenetic impacts of EDCs.
- Understanding these rapid signaling alterations is key to comprehending the full spectrum of EDC toxicity.
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