Role of mitochondria in tributyltin-induced interleukin-1alpha production in murine keratinocytes.
Tributyltin (TBT) salts are well known skin irritants in rodents and humans. TBT induced both the intracellular production of interleukin-1alpha (IL-1alpha) and its release into culture medium in a murine keratinocyte cell line (HEL30). Here, we report that mitochondria are important for TBT-induced IL-1alpha production. Confluent cells were treated with increasing concentrations of TBT (0--2.5 microM) 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-1alpha 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-1alpha synthesis induced by TBT, but rotenone did not completely abrogate TBT-induced IL-1alpha production, which suggests that other transcription factors may be involved in IL-1alpha 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-1alpha release was decreased by 65%, whereas no induction of intracellular IL-1alpha 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-(kappa)B activation by TBT. These findings indicate that mitochondria may be the source of second messenger molecules important for TBT-induced IL-1alpha production.
Corsini
E
E
Center for Cosmetic Toxicology, Institute of Pharmacologic Sciences, University of Milan, Italy.
Schubert
C
C
Marinovich
M
M
Galli
C L
CL
D000818:Animals; D002460:Cell Line; D007375:Interleukin-1 / Q000096:biosynthesis*; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism; D014220:Trialkyltin Compounds / Q000633:toxicity*
D007375:Interleukin-1; D016328:NF-kappa B; D014220:Trialkyltin Compounds; C011559:tributyltin