Role of Integrative Signaling Through Gap Junctions in Toxicology

Brad L. Upham1

1 Michigan State University, East Lansing, Michigan
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.18
DOI:  10.1002/0471140856.tx0218s47
Online Posting Date:  February, 2011
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Gap junctional intercellular communication (GJIC) plays a central role in coordinating signal‐transduction pathways that control gene expression inside of cells with those of neighboring cells in maintaining the homeostasis of a tissue. The normal homeostatic set point of gap junctions within tissues is in an open state, and although transient closure of gap junctions in response to mitogenic effectors is normal, chronic closure of channels by continuous exposure to environmental and food‐borne contaminants can result in adverse health effects such as cancer, teratogenesis, reproductive dysfunction, neuropathies, and cardiac arrhythmias. GJIC is the primary means of integrating signal transduction pathways controlling gene expression between contiguous cells. Thus, bioassay systems that can measure GJIC offer a central, more biosystems approach to assessing the potential for toxicants to epigenetically alter gene expression.Curr. Protoc. Toxicol. 47:2.18.1‐2.18.18. © 2011 by John Wiley & Sons, Inc.

Keywords: gap junctional intercellular communication (GJIC); scrape load‐dye transfer (SL‐DT); incision load‐dye transfer (IL‐DT); fluorescent recovery after photobleaching (FRAP); phosphatidyl choline specific phospholipase C (PC‐PLC); mitogen activated protein kinase (MAPK); extracellular receptor kinase (ERK); MAPK/ERK kinase1/2 (MEK1/2); reactive oxygen species (ROS)

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Table of Contents

  • Measurement of Gap Junctional Intercellular Communication
  • Strategic Planning
  • Basic Protocol 1: The Scrape‐Load Dye Transfer Technique
  • Basic Protocol 2: Quantitation of GJIC
  • Support Protocol 1: Determining Dose‐Response Conditions
  • Support Protocol 2: Determining Time‐Response and Time‐Recovery Conditions
  • Support Protocol 3: Determining GJIC Regulatory Mechanisms
  • Basic Protocol 3: In Vivo Analyses of Tumor‐Promoting and Chemopreventive Compounds
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: The Scrape‐Load Dye Transfer Technique

  • Monolayer of cells (F344‐WB from Drs. J.W. Grisham and M.S. Tsao, University of North Carolina‐Chapel Hill) cultured in D‐medium (formula no. 78‐5470EF, Invitrogen) supplemented with 5% FBS (Invitrogen), on 35‐mm tissue culture plates (Corning), and incubated at 37°C in a humidified 5% CO 2 incubator
  • Phosphate buffered saline (PBS; see recipe)or if cells tend to lift, use PBS with calcium and magnesium (see recipe).
  • 1.0 mg/ml Lucifer Yellow (LY) solution (see recipe)
  • 1.0 mg/ml LY/1.0 mg/ml rhodamine–dextran (RD) solution (see recipe; optional)
  • 10% (v/v) formalin (∼4% v/v formaldehyde; see recipe)
  • 35‐mm, 48‐well, or 96‐well cell culture plates
  • Surgical steel blade: no. 20 surgical blade and no. 4 handle; BD Bard‐Parker,
  • Epifluorescent phase contrast microscope or confocal microscope
  • 35‐mm camera or CCD camera

Basic Protocol 2: Quantitation of GJIC

  • Compounds to be tested for inhibition of GJIC

Support Protocol 1: Determining Dose‐Response Conditions

  • Stock solutions of U0126 (20 mM; see recipe), or the PC‐PLC inhibitor, D609 (20 mM; see recipe)
  • GJIC‐dysregulating chemical, e.g.: 1‐methylanthracene, 1‐methylfluorene, benzoylperoxide, 18‐β‐glycyrrhetinic acid, dicumylperoxide, fluoranthene, fluorene, pentachlorophenol, perfluorodecanoic acid, perfluorooctane sulfonic acid, phenanthrene, pyrene, 12‐O‐tetradecanoylphorbol‐13‐acetate (Sigma‐Aldrich), 1,9‐dimethylanthracene, 1‐methylpyrene, perfluorooctanoic acid, 2,2′,4,4′,5,5′‐hexachlorobiphenyl (PCB153) (Fluka), alachlor, lindane (Chem Service,, 1,1,1‐trichloro‐2,2‐di(4‐chlorophenyl)ethane (DDT; Supelco)

Support Protocol 2: Determining Time‐Response and Time‐Recovery Conditions

  • Liver of F344 rat treated with tumor‐promoting (see protocol 5) or chemopreventive compound (e.g., GJIC up‐regulators: resveratrol, β‐carotene, epigallocatechin gallate, retinoic acid, β‐sitosterol; Sigma‐Aldrich) (procedure can be adapted for other mammals)
  • 1.0 mg/ml Lucifer Yellow (LY; see recipe)
  • 10% (v/v) formalin (∼4% v/v formaldehyde; see recipe)
  • Plastic weigh plate
  • Gauze
  • Epifluorescent phase‐contrast microscope
  • Additional reagents and equipment for paraffin embedding (Zeller, ) and data analysis ( protocol 1)
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Literature Cited

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