Organotonin compounds such as trimethyltin (TMT) produce an unusual and specific spectrum of neurologic damage. Previous studies have demonstrated that a single peripheral dose of TMT produced a pattern of selective neurotoxicity. We have mapped the pattern of TMT toxicity in rat brain, finding destruction of selected population of neurons with no obvious interconnection. It was hypothesized that TMT-sensitive neurons expressed a gene product(s) that was responsible for the selection pattern of neurotoxicity. By using a combination of avidin/biotin subtractive hybridization and molecular cloning, we have isolated two cDNAs, named p9Tl9 and p9TlO, which appear to be expressed primarily in neurons destroyed by TMT as determined by in situ hybridization. Northern blot analysis demonstrated that p9Tl9 cDNA recognized an mRNA of 3.0 Kb, while p9TlO recognized a 2.6 Kb mRNA. Both mRNAs were not detected following TMT intoxication. In order to determine whether these cDNAs play a role in the mechanism of TMT toxicity, we propose the following experiments. Two near full-length clones for p9Tl9 have been isolated and will be sequenced; screening for a full-length clone corresponding to p9TlO is underway. The full sequence will be used to predict open reading frames, to generate primary amino acid sequences and to determine hydrophobicity plots. Synthetic peptides will be generated against selected regions of each open reading frame, and the resulting antisera will be used to characterize the gene products of these two cDNA clones. Both in situ hybridization using antisense p9Tl9 riboprobes and immunocytochemistry using the proposed antipeptide antisera will be performed on TMT-treated and untreated rats; both the mRNA and protein expression are expected to be greatly diminished in TMT-intoxicated rats. In order to determine whether expression of either cDNA confers TMT sensitivity in relatively non-sensitive cells, plasmids containing forward and reverse orientations of each cDNA driven by a constitutive CMV promotor will be constructed and transfected into transformed rat fibroblast, pheochromocytoma and human neuroblastoma cell lines. Once transfection and expression of the gene product is confirmed, sensitivity to the toxic effects of trimethyltin will be determined for transfected and control-transfected cell lines. If transfection of either cDNA confers sensitivity to trimethyltin in normally insensitive cell lines, we will have established a system in which to further analyze the mechanism of organotin toxicity. If structural analysis of the p9Tl9 gene product reveals any particular feature such as possible metal binding pockets which may account for the selective toxicity of TMT, it may be possible to determine whether toxic effects of other heavy metals such as lead are, in part, mediated by this gene product. By using the approach described in this proposal, we hope to establish a model paradigm for investigation of the molecular basis of selective organotin neurotoxicity, and to establish an in vitro system for analysis of the molecular mechanisms of such toxicity.
