Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
권호 표지
DOI QR코드

DOI QR Code

Optimization of Test Parameters and Interlaboratory Validation of Neuro-2a Assay for the Detection of Tetrodotoxin

테트로도톡신 검출을 위한 Neuro-2a 시험법의 시험 매개변수 최적화 및 실험실 간 검증 연구

  • Jeong-In Park (Department of Marine Science, Incheon National University) ;
  • Jun Kim (Department of Marine Science, Incheon National University) ;
  • Si-Yun Hong (Department of Marine Science, Incheon National University) ;
  • Youngjin Kim (Department of Marine Science, Incheon National University) ;
  • Hyun Park (Division of Biotechnology, Korea University) ;
  • Young-Seok Han (NEB Co.) ;
  • Youn-Jung Kim (Department of Marine Science, Incheon National University)
  • Received : 2024.10.21
  • Accepted : 2024.10.25
  • Published : 2024.10.30
Download PDF
⟨ Previous Next ⟩

Abstract

Tetrodotoxin (TTX) is a potent marine-derived neurotoxin. Existing detection methods for TTX, such as mouse bioassay (MBA) and LC-MS/MS, are limited by ethical concerns, and low detection thresholds, particularly in the absence of reference standards. Alternative testing methods are thus critically needed. The Neuro-2a assay is a well-established cell-based assay that uses mouse-derived Neuro-2a cells treated with ouabain (O) and veratridine (V), to induce cell death via excessive Na+ influx. This assay quantitates TTX based on its ability to inhibit Na+ influx, thereby allowing the cells to survive. In this study, we optimized parameters including TTX treatment conditions and O/V concentrations, to adapt the Neuro-2a assay for domestic laboratory conditions. The optimal O/V concentrations were determined to be 600/60 µM. We also identified eight concentration points (50-0.195 ng/mL) that generated a sigmoidal dose-response curve. By conducting 24 replicate experiments, we established six key data criteria to ensure reliability, with EC50 values ranging from 3.824 to 1.268 ng/mL. A comparison of inter-laboratory variability revealed that all quality control and data criteria values, except for COV+ and Bottom OD, showed coefficients of variation (CVs) ranging from 1.31 to 14.92%, confirming the assay's accuracy and reproducibility. In this study, we also elucidate optimal assay conditions, validated quality control and data criteria for TTX detection using the Neuro-2a assay in local laboratories. Additionally, we conducted an assay with 4,9-anhydroTTX, a TTX analog, and determined a TEF value of 0.2098, demonstrating the applicability of this method for detecting TTX and its analogs. This optimized Neuro-2a assay is expected to serve as an effective alternative to MBA for detecting TTX in domestic seafood products.

테트로도톡신(tetrodotoxin, TTX)은 강력한 해양생물 유래 신경독소로, 수산물 내 TTX를 검출하기 위해 기존에 주로 사용되는 mouse bioassay (MBA)와 LC-MS/MS 기법은 낮은 검출한계와 동물 윤리 문제 등의 한계가 있어 이를 대체할 새로운 시험법 개발이 필요합니다. Neuro-2a assay는 대표적인 세포기반 대체 시험법으로, 이 방법은 마우스 신경모세포인 Neuro-2a 세포주에 ouabain (O)과 veratridine (V)을 처리하여 과도한 Na+ 유입으로 인한 세포 사멸을 유도한 후, Na+ 채널 억제제인 TTX가 Na+ 유입을 차단해 세포를 보호하는 원리를 이용해 TTX를 정량합니다. 본 연구에서는 Neuro-2a assay를 국내 실험실 환경에 적용하기 위해 TTX 처리 조건과 O/V 농도 등의 매개변수를 최적화하였습니다. 그 결과, 최적 O/V 농도로 600/60 µM를 설정하였으며, S자형 용량-반응 곡선이 도출되는 8가지 농도(50-0.195 ng/mL)를 확인하였습니다. 또한, 24번의 반복 실험을 통해 데이터의 신뢰도를 평가할 수 있는 6가지 data criteria를 확립하였으며, 이 중 EC50 값은 약 3.824-1.268 ng/mL로 나타났습니다. 실험실 간 변동성 비교 결과, COV+와 Bottom OD값을 제외한 모든 품질 관리 기준(quality control criteria)과 데이터 기준(data criteria)의 변동계수(CVs)는 1.31-14.92%로 도출되어, 실험의 적정성과 재현성이 확인되었습니다. 본 연구는 국내에서 활용 가능한 TTX 검출용 Neuro-2a assay의 최적 조건과 신뢰성을 평가할 수 있는 quality control criteria와 data criteria를 제시하였습니다. 아울러, TTX뿐만 아니라 유사체인 4,9-anhydroTTX에 대한 TEF 값을 0.2098로 산출하여, TTX뿐 아니라 다양한 유사체의 검출이 가능함을 확인하였습니다. 향후, 본 시험법은 국내 수산물 내 TTX 검출을 위한 MBA 대체법으로 활용될 것으로 기대됩니다.

Keywords

Acknowledgement

본 연구는 2023도 식품의약품안전처의 연구개발비(20163MFDS641)로 수행되었으며, 이에 감사드립니다.

References

  1. Nam, K.T., Oh, S.J., Influence of water temperature and salinity on the production of paralytic shellfish poisoning by toxic dinoflagellate alexandrium catenella (Group I). J. Korean Soc. Mar. Environ. Saf., 27, 119-126 (2021). 
  2. Lee, K.J., Ha, K.S., Jung, Y.J., Mok, J.S.,Son, K.T., Lee, H.C., Kim, J.H., Paralytic shellfish toxins (PSTs) and tetrodotoxin (TTX) of Korean pufferfish. Fish. Aquat. Sci., 24, 360-369 (2021). 
  3. Kim, H.S., Kwun, H.J., Bae, H., Park, J., First reliable record of the blue-lined octopus, Hapalochlaena fasciata (Hoyle, 1886) (Cephalopoda: Octopodidae), from Jeju Island, Korea. J. Asia-Pac. Biodivers., 11, 21-24 (2018). 
  4. Kim, J.H., Suzuki, T., Shim, K.B., Oh, E.G., The widespread distribution of the venomous and poisonous blue-lined octopus Hapalochlaena spp., in the East/Japan Sea: possible effects of sea warming. Fish Aquat. Sci., 15, 1-8 (2012). 
  5. Kim, J.H., Kim, D.W., Cho, S.R., Lee, K.J., Mok, J.S., Tetrodotoxin and the geographic distribution of the blue-lined octopus Hapalochlaena fasciata on the Korean coast. Toxins, 15, 279 (2023). 
  6. Yasumoto, T., Yotsu-Yamashita, M., Chemical and etiological studies on tetrodotoxin and its analogs. J. Toxicol.: Toxin Rev., 15, 81-90 (1996). 
  7. Isbister, G.K., Kiernan, M.C., Neurotoxic marine poisoning. Lancet Neurol., 4, 219-228 (2005). 
  8. Bane, V., Lehane, M., Dikshit, M., O'Riordan, A., Furey, A., Tetrodotoxin: chemistry, toxicity, source, distribution and detection. Toxins, 6, 693-755 (2014). 
  9. Kim, J.H., Gong, Q.L., Mok, J.S., Min, J.G., Lee, T.S., Park, J.H., Characteristics of puffer fish poisoning outbreaks in Korea (1991-2002). J. Food. Hyg. Saf., 18, 133-138 (2003). 
  10. Ministry of Food and Drug Safety (MFDS), 2021. Standards and management measures for the detection of tetrodotoxin (TTX) in seafood, MFDS, Cheongju, Korea. 
  11. Japan Food Hygiene Association (JFHA), 1990. Standard methods of analysis in food safety regulation, biology, JFHA, Tokyo, Japan, pp. 86-87. 
  12. EFSA Panel on Contaminants in the Food Chain (CONTAM); Knutsen, H.K., Alexander, J., Barregard, L., Bignami, M., Bruschweiler, B., Ceccatelli, S., Cottrill, B., Dinovi, M., Edler, L., Grasl-Kraupp, B., Hogstrand, C., Hoogenboom, L., Nebbia, C.S., Oswald, I.P., Rose, M., Roudot, A.-C., Schwerdtle, T., Vleminckx, C., Vollmer, G., Wallace, H., Arnich, N., Benford, D., Botana, L., Viviani, B., Arcella, D., Binaglia, M., Horvath, Z., Steinkellner, H., van Manen, M., Petersen, A. Scientific opinion on the risks for public health related to the presence of tetrodotoxin (TTX) and TTX analogues in marine bivalves and gastropods. EFSA J., 15, 4752 (2017). 
  13. Park, B.K., Jang, W.J., Park, K.H., Lee, H.S., Lee, W.O., Choi, K.S., Lee, J., Tetrodotoxin and its Analogs: a review of analysis methods and levels in pufferfish. J. Food. Hyg. Saf., 36, 105-117 (2003). 
  14. Yasumoto, T., Fukui, M., Sasaki, K., Sugiyama, K., Determinations of marine toxins in foods. J. AOAC Int., 78, 574-581 (1995). 
  15. Kogure, K., Tamplin, M.L., Simidu, U., Colwell, R.R., A tissue culture assay for tetrodotoxin, saxitoxin, and related toxins. Toxicon, 26, 191-197 (1988). 
  16. Jellett, J.F., Marks, L.J., Stewart, J.E., Dorey, M.L., Watson-Wright, W., Lawrence, J.F., Paralytic shellfish poison (saxitoxin family) bioassays: automated endpoint determination and standardization of the in vitro tissue culture bioassay, and comparison with the standard mouse bioassay. Toxicon, 30, 1143-1156 (1992). 
  17. Castro, D., Manger, R., Vilarino, O., Gago-Martinez, A., Evaluation of matrix issues in the applicability of the neuro2a cell based assay on the detection of CTX in fish samples. Toxins, 12, 308 (2020). 
  18. Nicolas, J., Bovee, T.F., Kamelia, L., Rietjens, I.M., Hendriksen, P.J., Exploration of new functional endpoints in neuro-2a cells for the detection of the marine biotoxins saxitoxin, palytoxin and tetrodotoxin. Toxicol. in vitro, 30, 341-347 (2015). 
  19. Viallon, J., Chinain, M., Darius, H.T., Revisiting the neuroblastoma cell-based assay (CBA-N2a) for the improved detection of marine toxins active on voltage gated sodium channels (VGSCs). Toxins, 12, 281 (2020). 
  20. Aballay-Gonzalez, A., Ulloa, V., Rivera, A., Hernandez, V., Silva, M., Caprile, T., Delgado-Rivera, L., Astuya, A., Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples. Food Addit. Contam. Part A, 33, 869-875 (2016). 
  21. Aballay-Gonzalez, A., Gallardo-Rodriguez, J. J., Silva-Higuera, M., Rivera, A., Ulloa, V., Delgado-Rivera, L., Rivera-Belmar, A., Astuya, A., Neuro-2a cell-based assay for toxicity equivalency factor - proposal and evaluation in Chilean contaminated shellfish samples. Food Addit. Contam. Part A., 37, 162-173 (2020). 
  22. Kim, Y., Seo, J., Kim, J., Park, J.I., Kim, J.H., Park, H., Han, Y.S., Kim, Y.J., Establishment of test conditions and interlaboratory comparison study of neuro-2a assay for saxitoxin detection. J. Marine Life Sci., 9, 9-21 (2024). 
  23. Alkassar, M., Sanchez-Henao, A., Reverte, J., Barreiro, L., Rambla-Alegre, M., Leonardo, S., Mandalakis, M., Peristeraki, P., Diogene, J., Campas, M., Evaluation of toxicity equivalency factors of tetrodotoxin analogues with a neuro-2a cell-based assay and application to puffer fish from Greece. Mar. drugs, 21, 432 (2023). 
  24. Saruhashi, S., Konoki, K., Yotsu-Yamashita, M., The voltage-gated sodium ion channel inhibitory activities of a new tetrodotoxin analogue, 4,4a-anhydrotetrodotoxin, and three other analogues evaluated by colorimetric cell-based assay. Toxicon, 119, 72-76 (2016). 
  25. Yotsu-Yamashita, M., Urabe, D., Asai, M., Nishikawa, T., Isobe, M., Biological activity of 8,11-dideoxytetrodotoxin: lethality to mice and the inhibitory activity to cytotoxicity of ouabain and veratridine in mouse neuroblastoma cells, Neuro-2a. Toxicon, 42, 557-560 (2003). 
  26. Hamasaki, K., Kogure, K., Ohwada, K., A biological method for the quantitative measurement of tetrodotoxin (TTX): tissue culture bioassay in combination with a water-soluble tetrazolium salt. Toxicon, 34, 490-495 (1996). 
  27. Ledreux, A., Krys, S., Bernard, C., Suitability of the neuro-2a cell line for the detection of palytoxin and analogues (neurotoxic phycotoxins). Toxicon, 53, 300-308 (2009). 
  28. Caillaud, A., Eixarch, H., de la Iglesia, P., Rodriguez, M., Dominguez, L., Andree, K.B., Diogene, J., Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands. Food Addit. Contam. Part A, 29, 1000-1010 (2012). 
  29. Larsson, P., Engqvist, H., Biermann, J., Werner, R., Almlof, J., Johansson, F., Lundberg, E., Evers, K., Axen, M., Forssell-Aronsson, E., Kovacs, A., Karlsson, P., Helou, K., Parris, T.Z., Optimization of cell viability assays to improve replicability and reproducibility of cancer drug sensitivity screens. Sci. Rep., 10, 5798 (2020). 
  30. Van Dolah, F.M., Fire, S.E., Leighfield, T.A., Mikulski, C.M., Doucette, G.J., Determination of paralytic shellfish toxins in shellfish by receptor binding assay: collaborative study. J. AOAC Int., 95, 795-812 (2012). 
  31. Van Dolah, F.M., Leighfield, T.A., Doucette, G.J., Bean, L., Niedzwiadek, B., Rawn, D.F., Single-laboratory validation of the microplate receptor binding assay for paralytic shellfish toxins in shellfish. J. AOAC Int., 92, 1705-1713 (2009). 
  32. Diaz-Asencio, L., Clausing, R.J., Ranada, M.L., Alonso-Hernandez, C.M., Dechraoui Bottein, M.Y., A radioligand receptor binding assay for ciguatoxin monitoring in environmental samples: Method development and determination of quality control criteria. J. Environ. Radioact., 192, 289-294 (2018). 
  33. Nakamura, M., Yasumoto, T., Tetrodotoxin derivatives in puffer fish. Bull. Jpn. Soc. Sci. Fish., 51, 1341-1345 (1985).