Role of mitochondria in tributyltin-induced interleukin-1 alpha production in murine keratinocytes

Tributyltin (TBT) salts are well known skin irritants in rodents and humans, TBT induced both the intracellular production of interleukin-1 alpha (IL-1 alpha) and its release into culture medium in a murine keratinocyte cell line (HEL30), Here, we report that mitochondria are important for TBT-induced IL-1 alpha production. Confluent cells were treated with increasing concentrations of TBT (0-2.5 mu M) or dimethylsulfoxide as vehicle control, At different times thereafter (0-24 h), nuclear extracts were analyzed for nuclear factor-kappa B (NF-kappa B) binding activity by electrophoretic mobility shift assay, and the released and cell-associated IL-1 alpha was measured by enzyme-linked immunosorbent assay, TBT induced a direct and concentration-related activation of NF-kappa B, which peaked at 2 h and was blocked by pyrrolidinedithiocarbamate, a potent NF-kappa B inhibitor, and rotenone, an inhibitor of the electron entry from complex I to ubiquinone, Rotenone also induced a concentration-related inhibition of IL-1 alpha synthesis induced by TBT, but rotenone did not completely abrogate TBT-induced IL-1 alpha production, which suggests that other transcription factors may be involved in IL-1 alpha production. Prolonged treatment with ethidium bromide, an inhibitor of mitochondrial DNA and RNA synthesis, was used to partially deplete cells of functional mitochondria, After 5 d of treatment, mitochondrial conversion of tetrazolium bromide to formazan was reduced by 50%, and IL-1 alpha release was decreased by 65%, whereas no induction of intracellular IL-1 alpha was observed, This effect was not due to inhibition of protein synthesis, because identical incorporation of [H-3]leucine into protein in control and ethidium bromide-treated cells was identical, This impairment of mitochondrial metabolism inhibited NF-kappa B activation by TBT, These findings indicate that mitochondria may be the source of second messenger molecules important for TBT-induced IL-1 alpha production.