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Assessment of the Effects of Anatoxin-a In Vitro: Cytotoxicity and Uptake
Cristina Plata-Calzado1, Ana I Prieto1, Ana M Cameán1
1Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González 2, 41012 Seville, Spain.
Abstract:
Anatoxin-a (ATX-a) is a cyanotoxin whose toxicological profile has been underinvestigated in comparison to other cyanotoxins such as microcystins (MCs) or cylindrospermopsin (CYN). However, its wide distribution, occurrence, and toxic episodes justify more attention. It is classified as a neurotoxin, but it has also been reported to affect other organs and systems. Thus, the aim of this study was to establish, as a first tier in its toxicological evaluation, its cytotoxicity in a wide range of cell lines representative of potential target organs (N2a, SH-SY5Y, HepG2, Caco2, L5178Y Tk+/-, THP-1 and Jurkat). As limited effects were observed after exposure to up to 200 µg/mL of ATX-a for 24 h (only Jurkat and THP-1 cells showed reduced cell viability), cell uptake experiments were performed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The results showed that the immune system cells had the highest percentage of ATX-a in the intracellular fraction, followed by neuronal cells and finally Caco-2 and HepG2 cells. Moreover, the expression of genes related to cell death mechanisms in THP-1 cells was also analyzed by polymerase chain reaction (PCR) and showed no changes under the conditions tested. Further research is required on ATX-a's toxic effects and toxicokinetics to contribute to its risk assessment.
Insights
Anatoxin-a (ATX-a) shows limited cytotoxicity across most cell types. However, immune and neuronal cells accumulate higher levels of this cyanotoxin, warranting further investigation into its toxic effects.
Area of Science:
- Environmental Toxicology
- Cyanotoxin Research
- Cellular Toxicology
Background:
- Anatoxin-a (ATX-a) is an understudied cyanotoxin with potential impacts beyond neurotoxicity.
- Its widespread occurrence necessitates a better understanding of its toxicological profile.
- Previous research has not fully elucidated ATX-a's effects on various organs and systems.
Purpose of the Study:
- To evaluate the cytotoxicity of Anatoxin-a (ATX-a) in a diverse range of cell lines representing potential target organs.
- To investigate the cellular uptake of ATX-a in different cell types.
- To analyze gene expression related to cell death pathways following ATX-a exposure.
Main Methods:
- Cytotoxicity assays were performed on N2a, SH-SY5Y, HepG2, Caco2, L5178Y Tk+/-, THP-1, and Jurkat cell lines.
- Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to quantify intracellular ATX-a.
- Polymerase chain reaction (PCR) was employed to assess gene expression in THP-1 cells.
Main Results:
- Limited cytotoxicity was observed, with only Jurkat and THP-1 cells showing reduced viability at 200 µg/mL ATX-a after 24 hours.
- Immune system cells (THP-1, Jurkat) exhibited the highest intracellular ATX-a accumulation, followed by neuronal cells (N2a, SH-SY5Y).
- No significant changes in cell death-related gene expression were detected in THP-1 cells under the tested conditions.
Conclusions:
- Anatoxin-a demonstrates low cytotoxicity in most tested cell lines.
- Immune and neuronal cells show higher ATX-a uptake, suggesting potential target organ specificity.
- Further research into ATX-a's toxic effects and toxicokinetics is crucial for accurate risk assessment.
