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 that can comprehensively meet the demands for all toxicological assessments. Many in vitro assays are designed and optimized to evaluate as well as assess a specific biochemical or molecular endpoint. These are quite often combined in a high throughput set up to reflect a perturbation of a certain signal transduction pathway, such as estrogen-receptor signaling¹. This strategy has been quite successful, but the extensive number of signal transduction pathways involved in gene expression makes the task of choosing a specific signaling pathway to assess quite complex. High through-put protocols are currently being developed and used to simultaneously measure numerous signaling pathways, which has been one approach to overcome some of the limitations of single assays. However, not all signaling pathways have been successfully incorporated into more comprehensive approaches, plus new signaling pathways are constantly being discovered that further complicates this assessment process. Using extensive numbers of in vitro approaches, particularly high through-put systems, for comprehensive toxicological assessments are also very expensive and are not conducive to most single investigator led research projects.

GJIC is a process tightly controlled by changes in voltage, calcium concentration, pH, redox balance, regulated by major intracellular signal transduction pathways and interactions with membrane and cytoskeleton proteins^2,3. Thus, inhibition of GJIC can reflect different types of cellular stress, disruption of different cellular functions, or perturbations of different signal transduction pathways. Another approach to overcome the use of limited signal transduction bioassays is to take advantage of the biological phenomena that many, if not most, signal transduction pathways are further modulated by cooperative intercellular signaling systems through gap junction channels^4-8. Although intercellular signaling systems are also numerous and under multiple pathway control, the intercellular signaling through gap junction channels is ultimately a function of the channels being opened, partially closed or completely closed. This provides an endpoint that can be easily measured using various in vitro bioassay systems⁷. Considering that the homeostatic set point of a tissue requires open channels, determining the effect of compounds on gap junctional intercellular communication (GJIC) is a more comprehensive approach in determining potential toxic effects of compounds^4,8. In essence, this critical biological phenomenon that plays a central role in coordinating multiple signal transduction events controlling gene expression allows for a broad assessment of toxic effects. Thus, bioassays that assess GJIC are an excellent starting point to evaluate the toxic potential of compounds.

The most extensive techniques used to assess GJIC are based on preloading cells with a fluorescent probe and then monitoring the migration of the dye from the loaded cell or cells to adjacent cells. Techniques to preload the dye have involved microinjection⁹, scrape loading¹⁰, and methyl esters of the probes¹¹. The scalpel loading-fluorescent dye transfer (SL-DT) method is a modification of the scrape load - dye transfer assay developed by El Fouly¹⁰. Rather than the more invasive scrape, the scalpel loading method of this report involves a gentle roll of a scalpel with a round blade through a monolayer of cells that minimize invasive damage (Figure 1). The advantages of this technique are the toxicological assessment of a population of cells rather than single cells of the microinjection assay. Furthermore, the simplicity of this assay allows for rapid detection of multiple plates in a short time whereas methods using microinjection techniques and techniques that use methyl esters of fluorescent probes are significantly more time consuming and require considerably higher skill level.

Although there is no single method to meet all the needs of studying GJIC; the SL-DT assay is a simple, fairly inexpensive and versatile assay that can meet many of the needs for initial assessments of toxicities of various compounds. Major advantages include: simplicity, no special need for equipment or skills that are required for other methods such as microinjection, fluorescent recovery after photobleaching (FRAP) assay and local activation of molecular fluorescent probe assays, a rapid and simultaneous assessment of GJIC in a large number of cells, conducive to a high throughput set-up with automated fluorescence microscopy imaging and analysis, as well as its adaptability for in vivo studies.

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 the desired experimental treatment of the cells as described below.
   1. Conduct Dose Response Experiments
      1. Add 10 µl and 20 µl of a 2 mM stock solution of phenanthrene in 100 % acetonitrile and 4 µl, 6 µl, and 8 µl of a 20 mM stock solution of phenanthrene in 100 % acetonitrile to three plates of cells containing 2 ml of growth medium for each added volume of stock solution.
      2. Add 4 µl, 6 µl, 8 µl, 10 µl, and 20 µl of a 100% solution of acetonitrile to three plates of cells containing 2 ml of growth medium for each added volume of acetonitrile solution to serve as the vehicle control.
      3. Incubate plates for 15 min in an incubator set at 37 °C, 100% RH and 5% CO₂.
      4. Proceed to step 1.3.
   2. Conduct Time Response Experiments
      1. Add 7 µl of a 20 mM stock solution of phenanthrene in 100% acetonitrile to three plates of cells containing 2 ml of growth medium for each time point (i.e., 0, 1, 2, 3, 4, 5, 10 min).
      2. Add 7 µl of 100% acetonitrile to three plates of cells containing 2 ml of growth medium for each time point to serve as the vehicle control.
      3. Incubate plates for each designated time point in an incubator at 37 °C, 100% RH and 5% CO₂.
      4. Proceed to step 1.3.
   3. Conduct Time Recovery Experiments
      1. Add 7 µl of a 20 mM stock solution of phenanthrene in 100% acetonitrile to three plates of cells containing 2 ml of growth medium for 15 min.
      2. Add 7 µl of 100% acetonitrile to three plates of cells containing 2 ml of growth medium for 15 min to serve as the vehicle control.
      3. Decant the medium containing either the phenanthrene or acetonitrile and rinse 3x each with 3 ml of phosphate buffered saline (PBS).
      4. Add 2 ml of fresh growth medium to each plate and incubate for the desired recovery times (i.e., 25, 35, 45, 60, 90, 120, 150, 180, 240, 360 min) in an incubator at 37 °C, 100% RH and 5% CO₂.
      5. Proceed to step 1.3.
   4. Conduct Mechanism Experiments
      1. Add the signal transduction inhibitor, i.e., 5 µl of a 20 mM stock solution of D609 in 100% acetonitrile to three plates of cells containing 2 ml of growth medium for 15 min.
      2. Add 5 µl of 100% acetonitrile to three plates of cells containing 2 ml of growth medium for 15 min to serve as the vehicle control.
      3. Add 7 µl of a 20 mM stock solution of phenanthrene in 100% acetonitrile to three plates of cells containing 2 ml of growth medium plus the D609 for an additional 15 min.
      4. Add 7 µl of 100% acetonitrile to three plates of cells containing 2 ml of growth medium plus the 100% acetonitrile for an additional 15 min to serve as the vehicle control.
      5. Proceed to step 1.3.
3. Discard the culture medium by either gently pouring off the medium or by vacuum suction.
   Note: Dispose culture medium containing hazardous waste appropriately.
4. Rinse the cells three times each with 3 ml of PBS and either decant or aspirate between rinses.
5. Pipette 1 ml of 1 mg/ml Lucifer Yellow dye dissolved in PBS (LY) into each cell plate.
6. Preload the dye into the cells by gently rolling a #20 surgical steel blade with a rounded edge through a population of cells in three different areas of the plate.
   1. Begin by placing the scalpel perpendicular (90° angle) and 5 mm from the edge of the culture plate, and then roll the scalpel from this perpendicular angle to about an angle of 15° (see Figure 1).
      Note: This is achieved by pinching the scalpel between the index finger and thumb. Use gravity to allow the scalpel to gently go from the 90° to 15° position (as the rounded blade simply rolls over the cells). This motion will leave a visually noticeable indentation line.
7. Incubate the cells with the LY solution for 3 min at room temperature.
8. Decant or aspirate the LY and rinse three times with 3 ml of PBS to remove all extracellular dye to eliminate extracellular background fluorescence.
9. Add 0.5 ml of 10% phosphate buffered formalin solution to fix the cells.
   Note: After fixing in formalin, air dry the cells and store in the dark for up to two years with minimum photobleaching of the LY dye. When required, rehydrate with 10% formalin solution to visualize the fluorescent dye front.
10. View the fixed cells using an epifluorescence microscope equipped with a dichroic cube for excitation/emission peaks of 428/536 nm at a magnification of 200X. Align all plates so that the indentation line is parallel to the horizontal field of vision.
    Note: A FITC dichroic cube works well.
    1. Capture the image with a CCD camera and supporting software.
       1. Open the supporting software for the CCD camera and use the settings for auto exposure. Use the "capture" button to digitally acquire the image. Save the image as .tif files using the "Save" button.

2. Adaptation of the SL-DT Assay to Liver Tissue

1. Remove approximately a 2 x 2 cm slice from the left lobe of a liver from a 5 week old, male Fischer 344 rat using dissection scissors and place it on a plastic weigh plate covered with wet gauze¹².
2. Pipette 0.5 ml of a PBS solution containing 1 mg/ml each of lucifer yellow and rhodamine-dextran (RD) onto the surface of the liver slice in the plastic weigh plate.
   Note: The RD is a dye that cannot traverse gap junction channels and will mark the cells that were loaded.
3. Make three to five incisions approximately 1 cm long on the surface of the liver slice that has the dye solution in the weigh plate with a sharp blade and then add additional dye sufficient to fill the incisions.
4. Incubate the tissue for 3 min at room temperature on the plastic weigh plate.
5. Rinse three times with 5 ml of PBS.
6. Fix the tissue overnight in 30 ml of 10% phosphate buffered formalin in a 50 ml conical centrifuge tube in the dark at room temperature.
7. The following day, wash the slices with water, trim tissue around the incision with dissecting scissors into 1 x 1 x 0.5 cm³ strips and then use standard techniques to embed the slices in paraffin¹³.
8. Section the slices perpendicular to the incision line to thickness of 5 µm and store the samples in the dark until ready to be imaged using standard sectioning techniques with a microtome¹³.
9. Use an epifluorescence microscope equipped with a CCD digital camera to visualize and capture the images of the fluorescent dye fronts according to section 1.10⁷.

3. Quantifying GJIC

1. Measure the fluorescent dye spread using a morphometric software package (e.g., ImageJ).
   1. Click the "File" tab and then click "open" to open the saved image.
   2. Click on the "Free Hand Tool" in the Tool Bar Tab to trace the outline of the dye front.
   3. Click the "Analyze" tab and then click "Measure".
      Note: This will generate an area value in a spread sheet, which can be copied into other spread sheet programs if desired.
2. Using a spreadsheet program, compute the fraction of the control (FOC) by dividing the area of the dye spread in the experimental plate by the area the dye travelled in the control plate using the following equation:
   
   Ae = area of the dye spread in cells exposed to an experimental variable, such as cells treated with a chemical at a specific dose or time
   Ac = area of the dye spread in the control, which are cells treated with the vehicle used to dissolve the chemical of interest
   Note: To determine the effect of the vehicle, Ae would be the area of the dye spread in cells treated by the vehicle and Ac would be the area of the dye spread in cells not treated by the vehicle. The effect of the vehicle should preferably be less than 10%.

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/