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The Impact of O/V Content (Ouabain/Veratridine) and Matrix Effect on the Sensitivity of Ciguatoxin Detection in N2a Cytotoxicity Assay

學生姓名: 范馨月
指導教授: 陳泰源
學期: 113上
摘  要: Ciguatoxin (CTX) is a lipophilic marine neurotoxin that can cause gastrointestinal and neurological disorders in humans. Currently, research on CTX often employs a two-phase experimental approach. First, semi-quantitative bioassays (such as receptor binding assays and in vitro Neuro-2a cytotoxicity assays) are used to screen and analyze the toxicity in fish extracts. This is followed by chemical analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to confirm the presence and quantify the levels of CTX. In the Neuro-2a (N2a) cytotoxicity assay, sensitivity can be affected by factors such as the concentrations of ouabain and veratridine (O/V). Higher concentrations of O/V can cause greater cell damage, potentially masking positive results. Additionally, the lipids in the samples being tested can lead to matrix effects, resulting in non-specific toxic effects during the assay. Therefore, this study explores the impact of O/V concentration and matrix effects on the sensitivity of N2a cytotoxicity assays for detecting ciguatoxins. First, the study improved the specificity and sensitivity of in vitro detection of ciguatoxins (CTX) and brevetoxins (PbTx-3) by using N2a cell lines with lower sensitivity to O/V (OV-LS). The results showed that OV-LS cells could tolerate approximately twice the concentration of ouabain and veratridine compared to the original N2a cells, significantly enhancing the sensitivity to detect CTX3C, CTX1B, and PbTx-3. Next, by establishing the maximum tissue dose equivalent for different fish species with varying fat content and incorporating solid-phase extraction (SPE) purification procedures, matrix effects could be minimized without compromising sensitivity. The results indicated that after a twostep purification process (SPE Florisil and SPE C18), the non-specific toxicity caused by fish lipids on N2a cells was reduced.
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