@inproceedings{01H8KP5N0CNRDSZM6DMYWJMY7C,
  abstract     = {{Recently, the European Food Safety Authority (EFSA) published an external scientific report on the analysis of occurrence, exposure and toxicity of cyanobacteria toxins in food (Testai et al., 2017). For this type of food contaminants, microcystin-LR (MC-LR) is considered a significant member, garnering much attention due to its frequent detection and potent hepatotoxicity (Abdallah et al., 2021). The limited and
questionable nature of the current data pertaining to the prevalence of MC-LR in consumable animal byproducts, such as milk, makes it unfeasible to reach any definitive conclusion (Testai et al., 2017). On the other hand, aflatoxin M1 (AFM1) is a well-known fungal metabolite that contaminates milk, posing a
hepatic toxicity in human (Abdallah et al., 2019). Therefore, the current study aimed at exploring the
possible (co-)contamination of MC-LR and AFM1 in dairy milk samples and their combined toxicity on
HepG2 cells. An in-house LC-MS/MS method has been developed and validated for the detection of AFM1 and MC-LR in milk samples. The toxins are extracted by mixing the samples with 80% methanol followed by a filtration step using Bond Elut C18 cartridge for purification and clean-up. The method running time is 12 min, and the LOD and LOQ values are 0.3 ng/mL and 1 ng/mL for MC-LR and 0.015 ng/mL and 0.05 ng/mL for AFM1, respectively. The developed method is being applied to analyze several milk samples (n=113) that were randomly collected from different farms in Belgium during summer season. To determine the in vitro toxicity of both toxins, HepG2 cells have been exposed to MC-LR (1-500 ng/mL) and AFM1 (0.1-50 ng/mL) for 48 h. Neutral red uptake assay showed no cytotoxic effect up on the exposure to each toxin, however, a clear cytotoxic effect was observed at 500 ng/mL of MC-LR with either 25 or 50 ng/mL of AFM1. The bioenergetic status of the cells has been screened using Agilent Seahorse XFe96 Analyzer. Real-Time ATP Rate Assay kit and Cell Mito Stress Test kit were used to quantify the rate of ATP production (from glycolysis and mitochondrial respiration) and to assess the mitochondrial function, respectively. The results showed a significant mitochondrial toxicity, supported by a complete shutdown of ATP production, at 10 ng ng/mL of AMF1 and 100 ng/mL of MC-LR. Live-Cell analysis, using Incucyte® SX5 instrument, has been performed to study the apoptosis, mitochondrial membrane potential and adenosine triphosphate (ATP) metabolism, and the final results and conclusion will be presented during the EUROTOX 2023 conference.
Acknowledgment:
M.F.A holds a postdoctoral mandate funded by the Special Research Fund of Ghent University (BOF) - grant number BOF20/PDO/032. The Agilent Seahorse XFe96 Analyzer was purchased through the Special
Research Fund of Ghent University for GOA project no. 01G02213}},
  author       = {{Abdallah, Mohamed Fathi and Van Camp, Camille and Recote, Jessa May and Van Hassel, Wannes and Masquelier, Julien and Rajkovic, Andreja}},
  booktitle    = {{57th Congress of the European Societies of Toxicology (EUROTOX 2023), Abstracts}},
  issn         = {{0378-4274}},
  keywords     = {{Microcystin-LR,Aflatoxin M1,dairy milk,LC-MS/MS,Toxicology in vitro,HepG2,Mitochondrial toxicity}},
  language     = {{eng}},
  location     = {{Ljubljana, Slovenia}},
  number       = {{S1}},
  pages        = {{S230--S231}},
  title        = {{Microcystin-LR and aflatoxin M1 contamination in dairy milk samples and their combined in vitro hepatotoxicity}},
  url          = {{https://www.eurotox2023.com/}},
  volume       = {{384}},
  year         = {{2023}},
}