| Initial studies of this dissertation focused on the role of mitochondria-mediated apoptosis in environmentally related chemical toxicity and included a detailed characterization of S-(1,2-dichlorovinyl)-cysteine (DCVC)-induced mitochondrial dysfunction in cultured renal proximal tubular cells. DCVC is a potent nephrotoxicant and is a metabolite of trichloroethylene and dichloroacetylene, which are commonly encountered environmental pollutants. DCVC was shown to cause mitochondrial swelling, release of cytochrome c and activation of caspases in a Bcl-2 inhibitable manner. The data provide strong evidence that mitochondria-mediated apoptosis can be a central mechanism in environmental chemical toxicity.; The remaining studies focused on the function of a distinct form of thioredoxin (Trx) that is present in mitochondria, termed mtTrx or Trx2. The thioredoxin (Trx) family of proteins performs essential functions in protection against oxidative stress and regulation of apoptosis. To investigate the function of mtTrx, a putative human mitochondrial thioredoxin cDNA was cloned and studies were performed to determine if it functioned in mitochondria. Two mRNA transcripts were characterized and the transcription start site was identified. The protein was found to be widely distributed in different human tissues and the mRNA expression level correlates with the tissue mitochondria contents. Overexpression of mtTrx in 143B osteosarcoma cells protected against oxidantinduced apoptosis without affecting the clearance rate of peroxides.; In addition to protecting against chemical toxicity, inhibitory effects of Trx 1 on apoptosis may contribute to cancer formation. Measurement of mRNA levels for mtTrx in matched normal and tumor tissues showed that tumors had increased expression in colorectal and stomach cancer. Moreover, a thiol alkylating agent, N-ethylmaleimide (NEM), showed selective cytotoxicity in cells transfected with mtTrx at doses that did not affect vector-transfected cells. In contrast, cisplatin, a DNA crosslinking agent had no differential toxicity. The direct modification of mtTrx by NEM was shown by modified Redox Western blot approach. Together, the data indicate that mtTrx represents a potential target of cancer therapy. Significantly, the results suggest that targeting the conserved reactive sulfhydryl groups in the active center of Trx family proteins provides a new strategy for drug development. (Abstract shortened by UMI.)... |
