RESPIRATORY CARCINOGENESIS BY CHEMICAL AND PHYSICAL FACTORS

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Molecular markers and gene alterations were studied, using lung tissues from F344 rats treated with silica (12 mg Min-U-Sil 5 quartz), by single intratracheal instillation. This is a unique animal model for studying the pathogenesis of fibrosis-associated peripheral lung carcinomas. It yields a high incidence of peripheral lung carcinomas, derived from hyper- plastic alveolar type II epithelial cells, adjacent to silicotic granulo- mas. A study of TGF-beta1 distribution was completed (for rats observed for 6 months or longer), in silicotic granulomas and in adjacent alveolar type II hyperplasias, adenomas and carcinomas, using immunohistochemistry. Polyclonal antibodies to synthetic peptides, corresponding to the mature and precursor TGF-beta1, were localized as follows: TGF-beta1 precursor in the hyperplastic type II cells adjacent to silicotic granulomas, adenomatoid proliferations and adenomas, but not in carcinomas; mature TGF-beta1 was localized intracellularly in fibroblasts and macrophages at the periphery of silicotic granulomas adjacent to hyperplastic type II cells, and extracellularly in the connective tissue matrix adjacent to hyperplastic type II cells. These findings suggest a pathogenetic role for TGF-beta1 produced in the alveolar type II cells, both in silicosis and in associated carcinogenesis. Increased amounts of pan-reactive ras p21 protein were localized in hyperplastic alveolar type II cells, but not in adenomas and carcinomas. Immunohistochemical localization of p53 protein, using polyclonal antisera (CM-1) revealed nuclear reactivity in 25% of the carcinomas, but none in normal, hyperplastic or adenomatous type II cells. An RNA extraction method was developed for molecular analyses using paraffin embedded rat lung tissues with silica-associated carcinomas. Polymerase chain reaction on cDNA derived from extracted RNA, and also on extracted DNA, obtained from microdissected carcinomas, followed by single strand conformational polymorphism (SSCP) analysis and direct DNA sequencing, have so far showed no point mutations in exon 1 of the ras gene and exons 4-8 of the rat p53 gene.