Comparison of assays for gap junctional communication using human embryocarcinoma cells exposed to dieldrin.
Several assays were compared for their ability to detect inhibition of gap junctional communication in human embryocarcinoma cells exposed to the pesticide dieldrin. Included in this evaluation was a recently developed assay based on the transfer of the fluorescent dye derived metabolically from 5(and 6)-carboxy-2,7-dichlorofluorescein diacetate. This assay was compared to assays for fluorescence return after photobleaching (FRAP), transfer of Lucifer yellow after scrape-loading, autoradiographic visualization of [3H]-uridine nucleotide transfer, and metabolic coupling of 6-thioguanine (6-TG) metabolites. The scrape-loading assay was the most sensitive assay, detecting inhibition of junctional communication at all concentrations tested. Additionally, the scrape-loading assay provided the clearest demonstration of concentration-dependent inhibition of junctional communication, although the [3H]uridine assay also showed significant concentration-related effects. The 5(and 6)-carboxy-2,7-dichlorofluorescein diacetate and 6-TG metabolic coupling assays detected significant inhibition at the two highest concentrations of dieldrin only. The FRAP assay also detected substantial inhibition at the two highest concentrations only. These results show that scrape-loading is the most sensitive assay of those compared in this study for the detection of inhibited junctional communication. Furthermore, compared to the other assays evaluated, the newly developed 5(and 6)-carboxy-2,7-dichlorofluorescein diacetate assay is at least as sensitive, yet less cumbersome, less expensive, and more rapid. Finally, the results show that each of these assays was easily applied to embryonic cells, suggesting that they may be useful for evaluating disruption of junctional communication in embryonic cell cultures.
Loch-Caruso
R
R
Department of Environmental and Industrial Health, University of Michigan, Ann Arbor 48109-2029.
Caldwell
V
V
Cimini
M
M
Juberg
D
D
D001345:Autoradiography; D002450:Cell Communication / Q000187:drug effects*; D004396:Coloring Agents; D003584:Cytological Techniques; D004026:Dieldrin / Q000633:toxicity*; D005452:Fluoresceins; D005453:Fluorescence; D006151:Guanosine / Q000031:analogs & derivatives; D006801:Humans; D007365:Intercellular Junctions / Q000187:drug effects*; D013872:Thionucleosides; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism; D014500:Uracil Nucleotides / Q000378:metabolism
D004396:Coloring Agents; D005452:Fluoresceins; D013872:Thionucleosides; D014500:Uracil Nucleotides; D006151:Guanosine; C005783:6-thioguanosine; D004026:Dieldrin