Use of Neurite Outgrowth as an in Vitro Method of Assessing Neurotoxicity

Abstract

This work shows that the neurotoxic excitatory amino acids beta-N-methylamino alanine, BMAA, and kainate, modulate neurite outgrowth; this was assessed by measuring the levels of two separate neurofilament proteins (68 kD and 160 kD), in a mouse neuroblastoma cell line, (NB41A3). BMAA has been proposed to be the exogenous excitotoxin in Guam disease or amyotrophic lateral sclerosis (ALS/parkinsonian/dementia; Guam ALS-PD). Kainate is a glutamate analogue which causes excitotoxic damage associated with excessive entry of calcium into neurons. The results show that at low doses (10(-9) to 10(-7) M) both BMAA and kainate decrease the concentration of the two neurofilament proteins. However at high doses (10(-6) to 10(-5) M) they cause an apparent accumulation of the neurofilament proteins; the effect is more marked with BMAA. These results support the continued development of an in vitro test for neurotoxicity based on neurite outgrowth.

A Mathematical Model for Assessing Changes in Neurofilament Protein Levels in Neurites and Cell Bodies of Differentiating Neuroblastoma Cells

“…In the future, this model will be applied to studies on neurotoxicity testing, to determine how neurotoxicants a!ect the level of NF protein in di!erentiating SK-N-SH neuroblastomas. Previous studies have used ELISA to compare total NF protein levels in control and toxin-treated cells (Abdulla & Campbell, 1993). However, these studies did not address issues concerning the di!erent cell numbers (i.e.…”

“…In earlier studies in this lab, a simple in vitro neurotoxicity test was based on comparing total NF protein levels in control versus toxin-exposed cells after 6 days in culture (Abdulla & Campbell, 1993). This system, however, did not account for the fact that toxicants, in addition to altering neurite outgrowth, might also a!ect the total cell number (e.g.…”

“…During di!erentiation, the cells extend neurites and techniques such as image analysis can be used to obtain a direct measure of neurite length (Abdulla et al, 1995). Since neuro"lament (NF) proteins are major structural components of neurites, other studies have examined changes in the levels of NF proteins as potential indirect measures of neurite outgrowth (Doherty et al, 1984;Abdulla & Campbell, 1993): however, NF proteins are also present in the cell soma and the enzyme-linked immunoabsorbant assay (ELISA) used to assess these NF protein levels quanti"es the total NF protein in the cell population as a whole and does not separate the relative contribution made by the neurites and by the cell bodies. A mathematical model is presented to describe changes in the distribution of NF proteins within a cell during the process of neurite outgrowth.…”

Developmental neurotoxicity testing in vitro: Models for assessing chemical effects on neurite outgrowth

“…Neurofilament protein levels increase upon differentiation of cell lines (Yamazaki et al, 2005) and upon neurite outgrowth in primary cultures (Taniwaki and Schwartz, 1995). A correlation between chemical-induced reductions in neurite outgrowth and decreases in the level of neurofilament protein has been demonstrated in cell lines including NB41A3 cells (Abdulla and Campbell, 1993), SK-N-SH cells (Abdulla et al, 1995) and N2a cells (Flaskos et al, 2007). There may not be a direct relationship, however, between neurite length and neurofilament expression, as Cho and Tiffany-Castiglioni (2004) reported that decreased neurite length in SH-SY5Y cells in response to the pesticide exposure was associated with an upregulation of neurofilament protein.…”

Developmental Neurotoxicity of Anticholinesterase Pesticides

“…Indeed, in a number of cell culture studies a range of exogenous substances of widely differing structures have been shown to interfere with neurite outgrowth, although there is, so far, no evidence of those compounds being developmental neurotoxicants in vivo. These substances include the excitatory amino acids b-N-methylaminoalanine and kainate (Abdulla and Campbell, 1993), the antimalarial drug artemisinin (Smith et al, 1997), the insecticide pyrethrum (Axelrad et al, 2003), and many others. In fact, in several of the earlier studies, neurite outgrowth inhibition, instead of reflecting the potential of an OP to interfere with nervous system development, has been considered indicative of this compound's ability to cause neurodegenerative effects in vivo, especially in the context of the characteristic OP-induced delayed neuropathy (OPIDN) known to be elicited by some OPs (Flaskos et al, 1998;Henschler et al, 1992;Li and Casida, 1998;Schmuck and Ahr, 1997).…”