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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: December 25th, 2016

DOI:

10.3791/54281

1Department of Pediatrics & Human Development, Institute for Integrative Toxicology, Michigan State University, 2RECETOX — Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University

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.

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.

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 ....

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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.......

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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 .......

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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.......

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Supported by NIEHS grants #R01 ES013268-01A2, and the contents are solely the responsibility of the author and do not necessarily represent the official views of the NIEHS, and supported by CETOCOEN UPgrade project No. CZ.1.05/2.1.00/19.0382.

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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.
Image J National Institute of Health http://imagej.nih.gov/ij/

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