Abstract
In the last decades, the aquaculture industry has introduced plant-based ingredients as a source of protein in aquafeeds. This has led to mycotoxin contaminations, representing an ecological, health and economic problem. The aim of this study was to determine in the RTgill-W1 fish cell line the toxicity of fifteen mycotoxins of common occurrence in aquafeeds. To identify the most sensitive endpoint of toxicity, the triple assay was used. It consisted of three assays: alamarBlue, Neutral Red Uptake and CFDA-AM, which revealed the mitochondrial activity, the lysosomal integrity and the plasma membrane integrity, respectively. Most of the assayed mycotoxins were toxic predominantly at lysosomal level (enniatins, beauvericin, zearalenone, ochratoxin A, deoxynivalenol (DON) and its acetylated metabolites 15-O-acetyl-DON and 3-acetyl-DON). Aflatoxins B1 and B2 exerted the greatest effects at mitochondrial level, while fumonisins B1 and B2 and nivalenol were not toxic up to 100 µg/mL. In general, low toxicity was observed at plasma membrane level. The vast majority of the mycotoxins assayed exerted a pronounced acute effect in the fish RTgill-W1 cell line, emphasizing the need for further studies to ascertain the impact of mycotoxin contamination of fish feeds in the aquaculture industry and to establish safe limits in aquafeeds.
Highlights
The worldwide human population has experienced significant growth in the last decades, leading to an increasing demand for fish as an accessible protein source for human consumption [1,2,3]
The cytotoxic effects associated with different mycotoxins were determined in the RTgill-W1 cell line through the assessment of toxicity at mitochondrial level by the alamarBlue assay, at plasma membrane level by the carboxyfluorescein diacetate-acetoxymethyl ester (CFDA-AM) assay, and at lysosomal level by the neutral red uptake assay (NRU)
The effective concentrations that decreased mitochondrial activity or plasma or lysosomal membrane integrities in 50% of the cells (EC50 ) were calculated for each assay (Table 1). These values indicated a high toxicity for the ENNs, BEA and ZEN, which showed a greater effect at the lysosomal level with EC50 ranging from 2.89 to 8.02 μg/mL, the enniatin B (ENNB) being the least toxic (EC50 of 17.03 μg/mL)
Summary
The worldwide human population has experienced significant growth in the last decades, leading to an increasing demand for fish as an accessible protein source for human consumption [1,2,3]. Mycotoxins are toxic secondary metabolites produced by certain species of filamentous fungi that exhibit adverse effects in human and animals, leading to the development of various pathological conditions known as mycotoxicosis [1,8]. These conditions are very dissimilar in symptoms and severity, ranging from carcinogenicity to neurotoxicity, developmental toxicity and metabolic and reproductive disorders [1,4]
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