Loss of gap junctions from DDT-treated rat liver epithelial cells

Abstract

The mechanism by which the liver tumor promoter 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) inhibits gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells could involve gap junction loss and/or decreased gap junction channel permeability. We examined these two possibilities in the present study. Immunohistochemical studies using antibodies specific to connexin43, the major gap junction protein expressed by these cells, revealed that gap junction number and size were reduced during exposure to DDT. The reductions in gap junctions (33-91%) correlated with dose-dependent (1-10 microM) and time-dependent (0.5-4 h) decreases in cell-to-cell fluorescent dye-coupling (64-85%), as well as cellular levels of phosphorylated connexin43. These effects were reversible following removal of the tumor promoter from the culture medium, although cycloheximide reduced the level of gap junction reformation. The losses in gap junctions were not due to decreased connexin43 gene expression since steady-state levels of connexin43 mRNA were not similarly affected by DDT. Fenarimol (10 microM), a structural analog of DDT, did not inhibit GJIC and had no effect on gap junction structure or connexin43 expression. These data suggest that the inhibition of GJIC by DDT resulted from the removal of gap junctions from the plasma membrane and their degradation rather than simply a decrease in their permeability.

GCV phosphates are transferred between HeLa cells despite lack of bystander cytotoxicity

“…The variation in results between the two methods may be due to several factors. First, Lucifer Yellow microinjection relies on visual detection under a microscope of dye transfer, 47 whereas the double dye technique utilizes the more sensitive detection of fluorescence by flow cytometry. 46,48 Indeed, the flow diagrams indicated that there was measurable but low transfer of calcein in both cell types.…”

“…30,33 In addition, a double dye flow cytometry technique was utilized to measure GJIC during the 24-h period of GCV incubation used in the bystander cytotoxicity experiment compared to the brief 10 min evaluation period used for Lucifer Yellow microinjection. [46][47][48] The results demonstrated that, although Lucifer Yellow microinjection suggested there was no GJIC in U251 cells expressing the DNCx43 and in HeLa cells, GJIC was observed by the double dye flow cytometry method. Further studies investigated the role of low GJIC on GCV phosphate transfer and bystander cytotoxicity.…”

“…The multiple, slower migrating bands of the native Cx43 protein are due to different phosphorylation states. 47 Evaluation of GJIC by Lucifer Yellow microinjection demonstrated that GJIC was decreased in DNCx43-expressing cells (Table 1). To determine the effect of decreased GJIC on bystander cytotoxicity, three cell lines were chosen for further study to represent a range of GJIC: clones DN7, DN10, and DN14, which exhibited 52.1, 30.4, and 0% GJIC, respectively.…”

“…47 Frequency of dye transfer was determined visually. GJIC was measured as the percentage of directly adjacent cells that received dye from the microinjected central cell.…”

“…47 Briefly, a random primer-labeled [ 32 P]Cx43 cDNA was allowed to hybridize to the blots (15 mg total RNA per lane) overnight. The membranes were washed and exposed to Kodak XAR-5 X-ray film with an intensifying screen for 24 h at À801C, and then developed in an automatic X-ray film developer.…”