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Changes in ultrastructural characteristics and mucin gene expression were examined in rat tracheal explants cultured in a synthetic medium ± retinoic acid (RA), benzo[a]pyrene (B[a]P) and N-methyl-N-nitrosourea (NMNU). In the RA(+) cultures, no changes in either ultrastructural features or mucin gene expression were detected after 48h incubation. After 96h incubation, however, the ultrastructural features associated with the squamous phenotype were characteristics of cultures containing the two carcinogens and the mucin gene expression was slightly reduced. Thus, in the presence of retinoic acid, the carcinogen induced changes in cytology to the squamous phenotyps were not matched by a marked loss of mucin gene expression. Explants cultured for 48h without RA and±carcinogens showed none of the cytological changes associated with onset of the squamous phenotype. While mucin mRNA was still detected, it was clearly reduced compared to 48h cultures in RA(+) medium. However, 48h later, all explants exhibited pronounced squamous metaplasia and the mucin message decreased to trace levels. Thus, the results of these experiments with B[a]P and NMNU in RA(+) and RA(−) media indicates that at least the early carcinogen induced changes may be distinct from those associated with the retinoid pathway controlling expression of the mucin component of the mucociliary epithelium.