The effect of the cyanobacterial toxin saxitoxin on neurodevelopment

Abstract

The potent neurotoxin saxitoxin (STX) belongs to a group of structurally related analogues, collectively known as the paralytic shellfish toxins, produced by both marine and freshwater phytoplankton. This group of toxins act by blocking the voltage-gated sodium channels, halting the inflow of sodium ions and the subsequent generation of action potentials. While acute exposure has been well researched, with safety guidelines applied, chronic low dose exposure from neither marine or freshwater sources has been investigated. Given the role of cellular electrical activity in neurodevelopment this latter pattern of exposure may be of significant public health concern. This background has been addressed in chapter 1; “Low dose extended exposure to saxitoxin and its potential neurodevelopmental effects: a review”, and the published manuscript can be found in Appendix 1. Given this lack of investigation we aimed to determine if STX had an adverse effect on neurodevelopment following low dose extended exposure using two models of neuronal development. Further, we aimed to establish an assay which could be used to determine if any adverse neurodevelopmental effects recorded were due to direct STX toxicity. Firstly, using model neuronal cell lines it was shown that STX at or below the current drinking water guideline (0.25-3μg/L) caused a significant concentration dependent decrease in the development of neuronal morphology following an extended exposure period. This research is presented in chapter 2; “Extended low-dose exposure to saxitoxin inhibits neurite outgrowth in model neuronal cells” and the published manuscript can be found in Appendix 2. In addition to investigating the neurodevelopmental effects of STX, an assay measuring viability indirectly through cellular metabolism was established to be used with STX. The assay was used to eliminate the possibility of nonspecific cell toxicity as a cause of the effects on neurodevelopment. The assay was successfully optimised in two cell lines and tested with STX (0.25- 10μg/L) and ZnSO4 (10-4-10-1M), a known cytotoxic compound. The assay showed that STX is not toxic in our cell line under the conditions used for chapter 2. These results are reported in Chapter 3; “Optimisation of a real time resazurin based assay for use in OVCAR-3 and SH-SY5Y cells”. Moving to a model which more accurately models mammalian neuronal differentiation, the effect of STX at the drinking water guideline (3μg/L) and a predicted algal bloom concentration (10μg/L) was investigated using embryonic stem cells. Cells were differentiated using a previously described method of neuronal differentiation and assessed by examination of morphological development of neuronal features and expression of gene markers. A concentration dependent decrease in morphological neuronal index scores was recorded, confirming the results of chapter 1, in addition the expression of neuronal markers nestin and MAP2 were increased following exposure to STX (3μg/L) while ßIII-Tubulin was delayed by 3 days in both STX treatment groups. This research is presented in chapter 4; “Low dose exposure to saxitoxin affects neuronal differentiation of D3 embryonic stem cells”. These results suggest that STX, and potentially its analogues, interfere with proper neuronal development at environmentally relevant concentrations. Whilst further work is required to investigate the mechanisms causing the adverse effects seen, the work presented here raises awareness that this pattern of exposure could be of significant public health concern and deserves further investigation.