Down-regulation by butylated hydroxytoluene of the number and function of gap junctions in epithelial cell lines derived from mouse lung and rat liver

Abstract

The mouse pneumotoxicant and lung and liver tumor promoter butylated hydroxytoluene (BHT) was examined for its effects on gap junctional intercellular communication (GJIC) in mouse lung epithelial (C10) and rat liver epithelial (WB-F344) cell lines. GJIC, as measured by fluorescent 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 > or = 62.5 microM and in C10 cells at concentrations > or = 150 microM 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 also tested. In both cell lines BHTOH was a more potent inhibitor of GJIC than BHT, whereas BHTBzOH was ineffective. The mechanisms of inhibition of GJIC by BHT were also examined. The initial rapid inhibition detected within 15-30 min may have been due to gap junction channel closure or blockage, since no changes in gap junction number, connexin (Cx) 43 levels or Cx43 phosphorylation were observed. By 2-4 h, however, gap junctions were internalized into the cytoplasm, the number of immunodetectable plasma membrane gap junctions was reduced and phosphorylated 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 C10 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 epithelial cell GJIC through distinct mechanisms.

The vitamin E analog tocopherol succinate strongly inhibits gap junctional intercellular communication in rat liver epithelial cells (IAR203)

“…Second, the ability of TS to inhibit GJIC suggests that, at least in some conditions, it behaves like a tumor promoter [24,25]. Our results support epidemiological data suggesting the absence of a protective effect of vitamin E against cancer and even more its potential adverse effect: for example, a pooled analysis of prospective studies reported no protective effect of dietary vitamin E on lung cancer [48].…”

“…GJIC can be modulated by endogenous and exogenous agents. Most tumor promoters inhibit GJIC [24,25]; conversely, molecules having antipromoter activities can enhance GJIC [26,27].…”

Reduced connexin43 expression in high-grade human brain glioma cells

“…Reduction of gap junction communication often occurs at the promotion/progression stages of carcinogenesis. Many tumor promoters can disrupt gap junction communication (GJC) such as 12-O-tetradecanoylphorbol-13-acetate (TPA) [18,19], H 2 O 2 [20,21], okadaic acid [22] (our unpublished data) and butylated hydroxytoluene [23]. These data suggest that reduced expression of cx43 is related to tumor development rather than initiation of tumorigenesis.…”

Butylhydroxytoluene (BHT) increases susceptibility of transgenic rasH2 mice to lung carcinogenesis

“…(Witschi and Morse 1985). Recently, BHT has been reported also to induce inhibition of gap junctional intercellular communication and apoptosis in lung epithelial cells (Dwyer-Nield et al 1998;Guan et al 1995). Although any link between these events and the mechanisms underlying the tumorpromoting activity of BHT remains uncertain, it is likely that metabolites of BHT resulting from hydroxylation of a tert-butyl group by P450 isozymes play a key role in the chain of events leading to lung tumor development (Thompson et al 1989).…”