Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

Summary

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Abstract

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

Introduction

The overall goal of this method is to provide a simple, comprehensive and relatively inexpensive technique to assess the potential toxicities of compounds. This is an in vitro approach that can be used in multiple cell lines. Standard cell biology labs equipped with epifluorescence microscopes can conduct research using this assay.

Our basic knowledge of cell functions has been highly dependent on in vitro bioassays, and has become an essential component in toxicological assessments of pharmaceuticals, environmental pollutants, and food born contaminants. Unfortunately, there is no single in vitro bioassay system ....

Protocol

The protocol for this study was approved by the Animal Care and Utilization Committee of the National Institutes of Health Sciences of Japan, which is where the in vivo experiments were done, to assure that the rats were treated humanely and with regard for alleviation of suffering.

1. SL-DT Bioassay

1. Seed 3 x 10⁵ WB-F344 rat liver epithelial cells onto 35 mm diameter culture plates containing Eagles modified medium plus 5% fetal bovine serum, and culture the cells in an incubator at 37 ºC, 100% relative humidity (RH), 5% CO₂.
2. Culture the cells until they reach 100% confluence (typically, 2 days) and conduct t....

Results

Interruption of GJIC has been extensively used as a biomarker for identifying toxic compounds at the nongenotoxic, epigenetic level of gene control that induces adverse health effects¹⁴. For example, polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants of the environment but vary in their epigenetic toxicities as a function of their molecular structures¹⁵. The lower molecular weight PAHs are typically found at relatively higher concentrations than the .......

Discussion

The SL-DT assay is a simple and versatile technique in measuring GJIC, but there are several critical concerns that should be accounted for in designing appropriate experimental protocols. For robust measurements of GJIC using the SL-DT assay there must be a good dye spread of the LY through gap junctions of the cells. At minimum, an adequate time should be selected to assure that the dye spreads through eight or more rows of cells from the scalpel loaded cells in both directions. Also, for the ease of measurement of the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
WB-F344 rat liver epithelial cells	From Drs. J. W. Grisham and M. S. Tsao of the University of North Carolina (Chapel Hill, NC)	none	Provided by Drs. J. W. Grisham and M. S. Tsao University of North Carolina-Chapel Hill-NC
35 mm Culture Plates	Corning	430165
25 cm2 culture flasks	Corning	430639
75 cm2 culture flasks	Corning	430641
D-medium, an Eagles modified medium	ThermoFisher/GIBCO	Formula No. 78-5470EF
fetal bovine serum	ThermoFisher/GIBCO	10437
0.25% trypsin-EDTA	ThermoFisher/GIBCO	15050
phosphate buffered saline	homemade	see below for ingredient cat#'s	137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 2 mM KH2PO4
KCl	JT Baker - Mallinckrodt	3040-01
NaCl	JT Baker - Mallinckrodt	3624-05
Na2HPO4	JT Baker - Mallinckrodt	3819--01
KH2PO4	JT Baker - Mallinckrodt	3246-01
Lucifer Yellow CH, lithium salt	Sigma-Aldrich Chemical	L0259
rhodamine-dextran	Sigma-Aldrich Chemical	R9379
1-methylanthracene	Sigma-Aldrich Chemical
phenanthrene	Sigma-Aldrich Chemical	P11409
resveratrol	Sigma-Aldrich Chemical	R5010
D609	Tocris Bioscience	1437
acetonitril	EMD	AX0145-1
37% solution formaldehyde	JT Baker - Mallinckrodt	2106-01
#20 surgical blade	Fine Science Tools Inc.	10317-14
50 ml conical sterile tubes	Thermo scientific	339652
Nikon epifluorescence microscope	Nikon -Mager Scientific	Eclipse TE300
Nikon FITC dichroic cube	Nikon -Mager Scientific	96107
CCD camera	Nikon -Mager Scientific	Nikon Cool Snap EZ CCD
imaging system	Nikon -Mager Scientific	Nikon NIS-Elements F2.2 imaging system.
ImageJ	National Institute of Health	http://imagej.nih.gov/ij/