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Comparison of assays for gap junctional communication using human embryocarcinoma cells exposed to dieldrin
R Loch-Caruso1, V Caldwell, M Cimini
1Department of Environmental and Industrial Health, University of Michigan, Ann Arbor 48109-2029.
Abstract:
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.
Insights
The scrape-loading assay is the most sensitive method for detecting dieldrin
Area of Science:
- Cell Biology
- Toxicology
- Biochemistry
Background:
- Gap junctional communication is crucial for cellular function.
- Pesticides like dieldrin can disrupt intercellular communication.
- Assessing this disruption is vital for understanding toxic effects.
Purpose of the Study:
- To compare the sensitivity of various assays in detecting dieldrin-induced inhibition of gap junctional communication.
- To evaluate a new fluorescent dye assay for its efficacy and practicality.
Main Methods:
- Human embryocarcinoma cells were exposed to dieldrin.
- Assays compared included scrape-loading, fluorescence return after photobleaching (FRAP), [3H]-uridine nucleotide transfer, 6-thioguanine (6-TG) metabolic coupling, and a new 5(and 6)-carboxy-2,7-dichlorofluorescein diacetate assay.
- Inhibition of gap junctional communication was measured by dye transfer and metabolic coupling.
Main Results:
- The scrape-loading assay demonstrated the highest sensitivity, detecting inhibition at all dieldrin concentrations.
- Scrape-loading provided the clearest concentration-dependent inhibition data.
- The new fluorescent dye assay was as sensitive as other methods but faster and less expensive.
Conclusions:
- Scrape-loading is the most effective assay for detecting inhibited gap junctional communication.
- The new 5(and 6)-carboxy-2,7-dichlorofluorescein diacetate assay offers a practical alternative for evaluating junctional communication disruption.
- These assays are applicable to embryonic cell cultures for toxicity assessments.