Cytoskeletal changes in SY5Y neuroblastoma cells exposed to acrylamide: an immunocytochemical study

The neuronal cytoskeleton is vital for normal growth and differentiation of the nervous system, as well as for maintenance of the normal intracellular environment. Without it, major functional deficits occur due to interference with cellular transport of membrane components, proteins and neurotransmitter substances and as a result, inadequate maintenance of the distal axon occurs. Through the study of both nervous tissue and primary neuronal culture, specific cytoskeletal markers have been found to predominate in axonal or dendritic processes, as well as in different stages of neuronal development. In vitro study of neuroblastoma cell lines has also been utilized to develop hypotheses of neuronal development. These hypotheses attempt to explain the appearance of certain cytoskeletal elements, such as phosphorylated neurofilament proteins, in relationship to functional maturity of the neuron. We used SY5Y human neuroblastoma cells as an in vitro model of neurotoxicity to investigate cytoskeletal changes that may occur from the exposure of the nervous system to a known neurotoxicant. Cells were differentiated with either retinoic acid (RA) or dibutyryl cyclic adenosine monophosphate (dbcAMP) and 3-isobutyl-1- methyl-xanthine (IBMX). Differentiation was based morphologically on the appearance of neuritic processes in a majority (>50%) of the cells. Using the peroxidase-antiperoxidase technique, cells were labeled with monoclonal antibodies to cytoskeletal proteins (phosphorylated neurofilament, microtubule associated protein 2, vimentin and low molecular weight neurofilament protein) either specific for axonal markers or linked to stages in neuronal development. Staining patterns were compared to undifferentiated cells using the same protocol. There were no differences in staining patterns found between methods of differentiation or between differentiated cells and undifferentiated controls. Axonal markers of differentiation, defined as phosphorylated neurofilament immunopositivity, were only detected in cells exposed to retinoic acid for 9 days. Once these studies were completed, both differentiated and undifferentiated cultures were exposed to acrylamide as an example of a neurotoxicant with known cytoskeletal effects. Cells were fixed and stained after the observation of cellular swelling 24 hours post acrylamide treatment. In spite of obvious alterations in morphology in unstained cells in culture, the cytoskeletal staining pattern was unchanged after acrylamide treatment. We conclude that there is no difference in the cytoskeletal immunoreactivity of SY5Y neuroblastoma cells when differentiated with RA or dbcAMP/IBMX after three days. Retinoic acid differentiated cells, however, do develop immunoreactivity to axonal markers of differentiation after nine days of treatment. We also conclude that acrylamide does not affect the cytoskeletal structure of SY5Y neuroblastoma cells in undifferentiated or in RA or dbc AMP differentiated cells at the time and concentration tested.