Contribution of mass spectrometry to the advances in risk characterization of marine biotoxins: towards the characterization of metabolites implied in human intoxications

A significant spread and prevalence of algal toxins and, in particular, marine biotoxins have been observed worldwide over the last decades. Marine biotoxins are natural contaminants produced during harmful algal blooms being accumulated in seafood, thus representing a threat to human health. Significant progress has been made in the last few years in the development of analytical methods able to evaluate and characterize the different toxic analogs involved in the contamination, Liquid Chromatography coupled to different detection modes, including Mass Spectrometry, the method of choice due to its potential for separation, identification, quantitation and even confirmation of the different above-mentioned analogs. Despite this, the risk characterization in humans is still limited, due to several reasons, including the lack of reference materials or even the limited access to biological samples from humans intoxicated during these toxic events and episodes, which hampered the advances in the evaluation of the metabolites responsible for the toxicity in humans. Mass Spectrometry has been proven to be a very powerful tool for confirmation, and in fact, it is playing an important role in the characterization of the new biotoxins analogs. The toxin metabolization in humans is still uncertain in most cases and needs further research in which the implementation of Mass Spectrometric methods is critical. This review is focused on compiling the most relevant information available regarding the metabolization of several marine biotoxins groups, which were identified using Mass Spectrometry after the in vitro exposition of these toxins to liver microsomes and hepatocytes. Information about the presence of metabolites in human samples, such as human urine after intoxication, which could also be used as potential biomarkers for diagnostic purposes, is also presented

Evaluation of matrix issues in the applicability of the neuro-2a cell based assay on the detection of CTX in fish samples

Ciguatoxins (CTXs) are a group of neurotoxins responsible for the syndrome ciguatera fish poisoning (CFP) as a result of the consumption of contaminated fish. The presence of these toxins has been detected around the Pacific, Caribbean and Indian coasts. Recent reports indicate the emergence of CFP in other geographic areas, in particular in European coasts, of the Canary Islands (Spain) and Madeira (Portugal). A neuroblastoma cell line of murine origin (N2a) has been applied to assay different groups of neurotoxins, acting on voltage-gated sodium channel (VGSC) of excitable cells, N2a-MTT. The great potential of N2a-MTT as a sensitive tool for the CTXs screening is clearly recognized, notably because it allows the detection of these toxins at levels below recommended as security levels. However, the complexity of the matrix is a critical point on the application of N2a-MTT, which needs to be evaluated. The aim of this work is to provide recommendations for an implemented N2a-MTT method for CTXs determination in fish that avoids matrix effects, particularly those related to high lipid content.European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/0

Melatonin content in walnuts and other commercial nuts. Influence of cultivar, ripening and processing (roasting)

Nuts are important components of a healthy diet since they provide nutritional value and bioactive components. Melatonin is a well-known molecule in plants, and its relevance in foodstuffs is increasing. This study investigated the presence of melatonin in nuts using chromatographic techniques and optimized extraction procedures according to the high oil content of nuts. Melatonin was detected in four walnut cultivars with levels similar to those previously reported. Moreover, the melatonin content in walnut seeds decreased sharply during the ripening process from the ripe green stage to the mature dehydrated fruit and increased after harvesting when the fruits were edible. A number of other commercial nuts were also measured, with melatonin contents varying markedly, and generally being lower than in walnuts. The presence of melatonin was lower in commercial roasted nuts than in raw nuts, with the exception of peanuts, where melatonin content increased with roasting. It seems that this industrial processing negatively alters the structure of this molecule and its availability, which should be taken into account when estimating its levels during nut consumption. Therefore, this study reveals new data on the presence of melatonin in walnut seeds in a natural format and its evolution with maturation as well as in other commercial nuts. We also highlight the importance that processing has on melatonin and other antioxidants in the nuts that reach the consumer.Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGXunta de Galicia | Ref. ED481A-2017/38

Preparation of ciguatoxin reference materials from Canary Islands (Spain) and Madeira Archipelago (Portugal) fish

Ciguatoxins (CTXs) are naturally occurring neurotoxins that can accumulate in fish and cause Ciguatera Poisoning (CP) in seafood consumers. Ciguatoxic fish have been detected in tropical and subtropical regions of the world including the Pacific and Indian Oceans, the Caribbean Sea, and more recently in the northeast Atlantic Ocean. The biogeographic distribution of ciguatoxic fish appears to be expanding; however, the paucity of CTX standards and reference materials limits the ability of public health authorities to monitor for these toxins in seafood supply chains. Recent reports establish that Caribbean Ciguatoxin-1 (C-CTX1) is the principal toxin responsible for CP cases and outbreaks in the northeast Atlantic Ocean and that C-CTX congener profiles in contaminated fish samples match those from the Caribbean Sea. Therefore, in this work, C-CTX reference materials were prepared from fish obtained from the northeast Atlantic Ocean. The collection of fish specimens (e.g., amberjack, grouper, or snapper) was screened for CTX-like toxicity using the in vitro sodium channel mouse neuroblastoma cytotoxicity assay (N2a cell assay). Muscle and liver tissues from toxic specimens were pooled for extraction and purified products were ultimately profiled and quantified by comparison with authentic C-CTX1 using LC-MS/MS. This work presents a detailed protocol for the preparation of purified C-CTX reference materials to enable continued research and monitoring of the ciguatera public health hazard. To carry out this work, C-CTX1 was isolated and purified from fish muscle and liver tissues obtained from the Canary Islands (Spain) and Madeira archipelago (Portugal).European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/03Xunta de Galicia | Ref. ED481A-2018/20