DOWN-REGULATION BY BUTYLATED HYDROXYTOLUENE OF THE NUMBER AND FUNCTION OF GAP-JUNCTIONS IN EPITHELIAL-CELL LINES DERIVED FROM MOUSE LUNG AND RAT-LIVER

The mouse pneumotoxicant and lung and liver tumor promoter butylated h ydroxytoluene (BHT) was examined for its effects on gap junctional int ercellular communication (GJIC) in mouse lung epithelial (C10) and rat liver epithelial (WB-F344) cell lines, GJIC, as measured by fluoresce nt dye microinjection, was inhibited in both types of cells by BHT in dose- and time-dependent fashions, Inhibition was detected in WB-F344 cells at BHT concentrations greater than or equal to 62.5 mu M and in C10 cells at concentrations greater than or equal to 150 mu M after 4 h treatment, Inhibition occurred within 15-30 min and was reversed by removing BHT from the culture medium, The highly toxic BHT metabolite 6-t-butyl-2-(hydroxy-t-butyl)-4-methylphenol (BHTOH) and the non-toxic BHT metabolite, 2,6-di-t-butyl-4-hydroxymethylphenol (BHTBzOH) were a lso tested, In both cell lines BHTOH was a more potent inhibitor of GJ IC than BHT, whereas BHTBzOH was ineffective, The mechanisms of inhibi tion of GJIC by BHT were also examined. The initial rapid inhibition d etected within 15-30 min may have been due to gap junction channel clo sure or blockage, since no changes in gap junction number, connexin (C x) 43 levels or Cx43 phosphorylation were observed, By 2-4 h, however, gap junctions were internalized into the cytoplasm, the number of imm unodetectable plasma membrane gap junctions was reduced and phosphoryl ated Cx43-P2 was decreased, Treatment of the cells for 24 h with 12-O- tetradecanoylphorbol-13-acetate (TPA) prevented inhibition of GJIC by TPA, but not by BHT. Western blot analyses of TPA-treated WB-F344 or C 10 cells revealed the presence of a hyperphosphorylated form of Cx43 ( Cx43-P3) and no reduction in Cx43-P2, in contrast to BHT-treated cells , These data suggest that BHT and TPA inhibit lung and liver epithelia l cell GJIC through distinct mechanisms.