Abstract
Alternaria mycotoxins are secondary fungal metabolites which can contaminate food and feed. They are produced by Alternaria species with alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and tentoxin (TEN) as the main representatives for Alternaria mycotoxins in food. Once passing the intestinal barrier, Alternaria toxins can reach the liver to exert yet uncharacterized molecular effects. Therefore, hepatic in vitro systems were used to examine selected Alternaria mycotoxins for their induction of metabolism-dependent cytotoxicity, phosphorylation of the histone H2AX as a surrogate marker for DNA double-strand breaks, and relevant marker genes for hepatotoxicity. Analysis of cell viability as well as the induction of H2AX phosphorylation in the hepatocarcinoma cell line HepG2 revealed a detoxification of 100 μmol/l AME and AOH by pre-treatment with S9 liver homogenate as shown by a decrease in cytotoxicity and H2AX histone phosphorylation to levels observed in control cells. Concentrations up to 100 μmol/l TeA and TEN did not induce H2AX phosphorylation whether metabolized or not. In the metabolically competent human hepatoma cell line HepaRG, no cytotoxicity of Alternaria toxins occurred even at high concentrations up to 100 μmol/l, which indicates a low cytotoxic potential. Induction of gene expression associated with liver toxicity was analyzed by quantitative real-time PCR using a specific hepatotoxicity PCR array in HepaRG cells: here, an evidence was found that 50 μmol/l of AOH, AME, TeA, and TEN might be associated with hepatotoxic effects, necrosis, and the development of diseases like cholestasis and phospholipidosis.
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Abbreviations
- AOH:
-
Alternariol
- AME:
-
Alternariol monomethyl ether
- b. w.:
-
Body weight
- CYP:
-
Cytochrome P450 monooxygenase(s)
- MTT:
-
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
- OA:
-
Okadaic acid
- TEN:
-
Tentoxin
- TeA:
-
Tenuazonic acid
- UGT:
-
UDP-glucuronosyltransferase(s)
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Acknowledgements
We would like to thank Horst Stefan Klaffke for the inspiring and motivating discussions and Corinna Genrich for her excellent technical support.
Funding
This work was supported by the German Federal Institute for Risk Assessment (grant numbers 51-006 and 1322-623).
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Experiments with animals for preparing rat S9 fraction were performed according to European laws and with consent of the responsible authority of the state of Berlin (Regional Office for Health and Social Affairs Berlin—LAGeSo), numbers H0256/02 (treatment of animals) and T0258/02 (killing of animals and removal of organs).
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Hessel-Pras, S., Kieshauer, J., Roenn, G. et al. In vitro characterization of hepatic toxicity of Alternaria toxins. Mycotoxin Res 35, 157–168 (2019). https://doi.org/10.1007/s12550-018-0339-9
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DOI: https://doi.org/10.1007/s12550-018-0339-9