EFFECTS OF ETHYLNITROSOUREA ON EXPRESSION OF PROTOONCOGENE PP60(C-SRC) AND HIGH-MOLECULAR-WEIGHT NEUROFILAMENT PROTEIN IN RODENT EMBRYO CENTRAL-NERVOUS-SYSTEM CELLS IN-VITRO

Effects of exposure to ethylnitrosourea (ENU) on expression of proteins that play a role in neuronal differentiation were examined in central nervous system (CNS) micromass embryo cell cultures. ENU is a known developmental toxicant which affects neuronal development. The proteins selected were the protein product of the src proto-oncogene (pp60(c-src)) and high-molecular-weight neurofilament protein (NF). pp60(c-src) has marked increases in amount and kinase activity in neurons at the time of differentiation and NF is found in differentiated neurons. CNS micromass cultures are primary cells from Day 12 rat embryo midbrains which are plated at high density and differentiate into neurons during 5 days in culture. Proteins were quantitated by polyacrylamide gel electrophoresis separation of equal amounts of total cell protein followed bq transfer to membranes, immunoblotting, and densitometric scanning of blots. Dose-dependent decreases in cell growth and differentiation were confirmed using endpoints of cell number, protein content of cultures, neutral red uptake, hematoxy lin staining of differentiated cells, and levels of binding of the neurotransmitter [H-3]gamma-aminobutyric acid. Concentrations,which inhibited response by 50% compared to controls ranged from 232 to 455 mu M ENU. Dose-related decreases in amounts of pp60(c-src) and NF proteins relative to total protein were seen in CNS cultures treated with ENU. Results confirm the usefulness of the micromass cultures in following chemical effects on neuronal differentiation. The effects of ENU on specific proteins associated with neuronal differentiation were shown. (C) 1994 Academic Press, Inc.