Trimethyltin (TMT) is selective toxicant, causing neurotoxicity, nephrotoxicity and immunotoxicity. In this proposal, we plan to test whether TMT toxicity is caused by the specific expression of a protein, termed stannin, in TMT-sensitive tissues. Stannin was isolated using subtractive hybridization of toxicant-treated brains and molecular cloning techniques. A full-length cDNA was isolated and encoded novel, 10 kDa peptide; this was verified using in vitro transcription and translation. Antipeptide antisera, in situ hybridization and/or Northern blots indicated that TMT-sensitive neurons, spleen and kidney cells expressed stannin. In vitro analysis showed that neuroblastoma lines were sensitive to TMT, whereas glioma and fibroblasts were not; transfection studies are in progress. Stannin was expressed as a stannin fusion proteins in bacteria. Bacteria expressing the stannin fusion were sensitive to TMT, suggesting that expression of stannin is sufficient to confer TMT sensitivity. If stannin expression alone is necessary and sufficient for TMT toxicity, transfected or transgenic, TMT-insensitive cells and tissues should -become sensitive to TMT. This renewal is focused on several areas. 1) We propose to determine whether stannin can be used as a biomarker for exposure to metal toxicants, and will develop immunoassays and PCR-based assays for testing toxicant-exposed tissues and body fluids. 2) The biologic and biophysical properties of stannin will be determined using antisera and stannin fusion proteins. Stannin will be cleaved from the fusion protein, and used to determine if TMT or other metals bind to the protein. 3) Cell culture systems will be used to characterize stannin-TMT interactions, using antisense oligonucleotides to block stannin expression. 4) Several putative human genomic clones for stannin were isolated; stannin gene structure and chromosomal location will be determined. 5) Transgenic mice will be developed with stannin gene expression directed to tissues which do not express stannin; such tissues include pancreas via elastase promoter expression, and cerebellar Purkinje cells via the L7 promotor. Ultimately, targetted gene disruptions may be used to see if stannin-deficient mice are insensitive to TMT. Stannin expression in heterologous tissues of transgenic mice may provide convincing evidence that stannin confers TMT sensitivity, and will allow development of transgenic strains for use in other aspects of neurotoxicity.
