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

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