IN VITRO RESPIRATORY TOXICITY SCREENING TESTS

WE PROPOSE TO ESTABLISH PERMANENT, FUNCTIONALLY DIFFERENTIATED GUINEA PIG RESPIRATORY EPITHELIAL CELL LINES FOR USE IN THE DEVELOPMENT OF SHORT-TERM IN VITRO RESPIRATORY TOXICITY SCREENING TESTS. TO DO THIS, WE PROPOSE TO DETERMINE THE CULTURE CHARACTERISTICS OF GUINEA PIG RESPIRATORY EPITHELIAL CELLS AND TO EVALUATE THEM FOR THEIR EXPRESSION OF THREE CHARACTERISTIC DIFFERENTIATED PHENOTYPES: MUCIN PRODUCTION; CYTOCHROME P450 INDUCTION; AND FINE MORPHOLOGY. PRIMARY CELL POPULATIONS THUS CHARACTERIZED WILL BE EMPLOYED IN STUDIES AIMED AT EXTENDING THEIR STABILITY AND GROWTH PARAMETERS. TWO STRATEGIES WILL BE EXPLOITED: REVERSIBLE TRANSFORMATION WITH THE IMOORTALIZING AND TRANSFORMING LARGE T ANTIGEN ONCOGENE OF SV40, UNDER THE CONTROL OF THE REGULABLE MOUSE METALLOTHIONEIN PROMOTER AND/OR STABLE IMMORTALIZATION WITH THE "IMMORTALIZING" BUT NOT "TRANSFORMING" ELA ONCOGENE OF ADENOVIRUS, UNDER CONSTITUTIVE, NONREGULABLE CONTROL OF THE SV40 PROMOTER AND RETROVIRAL LONG TERMINAL REPEATS (LTS'S). TRANSFORMED, IMMORTALIZED RESPIRATORY CELL LINES THUS OBTAINED WILL BE EVALUATED FOR THEIR EXPRESSION OF THE RESPIRATORY EPITHELIAL DIFFERENTIATED PHENOTYPE BY USE OF COMPARATIVE ANALYSES VS. PRIMARY POPULATIONS.