Abstract
Alternaria molds produce a variety of chemically diverse secondary metabolites with potentially adverse effects on human health. However, data on occurrence in food and human exposure is inconsistent for some of these mycotoxins. Membrane filtration is a frequent step in many sample preparation procedures for LC-MS-based methods analyzing food contaminants. Yet, little is known about the possibility of adsorptive phenomena that might result in analyte losses. Thus, we treated a complex extract of Alternaria toxins with several types of syringe filters and unraveled the impact on its chemical composition by LC-MS/MS. We observed significant, and in some cases complete, losses of compounds due to filtration. Particularly, two key Alternaria toxins, alternariol (AOH) and its monomethyl ether (AME), were heavily affected. As a comparison with published food surveys indicating a correlation of the type of filtration used with lower incidence reports in food, our results point at a possible underestimation of AME in past exposure assessment. Also, perylene quinones were greatly affected by filtration, underlining the importance to take this into consideration during analytical method development. Furthermore, we applied the comet assay in HT-29 cells to elucidate the impact of filtration on the genotoxicity of the extract. We observed strong coincidences with the loss of epoxide-carrying metabolites and also an intriguing induction of oxidative DNA damage by yet toxicologically uncharacterized Alternaria toxins. In conclusion, we highlight potential issues with sample filtration and call for a critical re-evaluation of previous food occurrence data in the light of the results at hand.
Highlights
Black molds of the genus Alternaria are ubiquitous microorganisms, which can grow on a variety of substrates, such as soil, decaying organic materials, and agricultural crops
A prominent difference between the two compounds appeared with polyvinylidene difluoride (PVDF) membranes, as it adsorbed alternariol monomethyl ether (AME) to a much higher extent as compared with AOH (24% reduction)
Syringe filters are widely applied in sample preparation for LC-MS based food safety surveys, yet little is known about possible interactions between the membrane material and potential analyte losses
Summary
Black molds of the genus Alternaria are ubiquitous microorganisms, which can grow on a variety of substrates, such as soil, decaying organic materials, and agricultural crops. Two toxins belonging to the group of dibenzo-αpyrones, namely alternariol (AOH) and alternariol monomethyl ether (AME), are considered a higher risk due to their potential impact on DNA integrity Both toxins are very similar in chemical structure (Fig. 1) and biological activity. They are known to target human topoisomerase II, leading to DNA doublestrand breaks in vitro (Fehr et al 2009) They were reported to modulate inflammatory responses (Kollarova et al 2018; Solhaug et al 2015) and to interact with steroid receptors (Dellafiora et al 2018; Frizzell et al 2013; Lehmann et al 2006; Stypuła-Trębas et al 2017; Vejdovszky et al 2017). The reported prevalence in food samples, the EFSA CONTAM panel lately estimated the 95th percentile dietary exposure of the European population to exceed toxicological threshold of concern (TTC) values (EFSA 2016)
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