Gut microbiota and undigested food constituents modify toxin composition and suppress the genotoxicity of a naturally occurring mixture of Alternaria toxins in vitro

Francesco Crudo,Jovana Mihajlovic,Chiara Dall'Asta,Hannes Puntscher,Elisabeth Varga,David Berry,Georg Aichinger,Doris Marko,Luca Dellafiora,Benedikt Warth

doi:10.1007/s00204-020-02831-1

Francesco Crudo, Jovana Mihajlovic + Show 8 more

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https://doi.org/10.1007/s00204-020-02831-1

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Abstract

Molds of the genus Alternaria produce several mycotoxins, some of which may pose a threat for health due to their genotoxicity. Due to the lack of adequate toxicological and occurrence data, they are currently not regulated. Interactions between mycotoxins, gut microbiota and food constituents might occur after food ingestion, modifying the bioavailability and, therefore, overall toxicity of mycotoxins. The present work aimed to investigate the impact of in vitro short-term fecal incubation on the in vitro DNA-damaging effects exerted by 5 µg/mL of an Alternaria alternata extract, containing, among others, 15 nM alternariol, 12 nM alternariol monomethyl ether, 241 nM altertoxin II and 301 nM stemphyltoxin III, all of which are known as genotoxic. The involvement of microorganisms, undigested food constituents and soluble substances of human fecal samples in modifying the composition and the genotoxicity of the extract was investigated through the application of LC–MS/MS analysis and comet assays in HT-29 cells. Results showed that the potential of the mycotoxins to induce DNA strand breaks was almost completely quenched, even before anaerobic incubation, by contact with the different fractions of the fecal samples, while the potency to induce formamidopyrimidine DNA glycosylase (FPG)-sensitive sites was only slightly reduced. These effects were in line with a reduction of mycotoxin concentrations found in samples analyzed by LC–MS/MS. Although a direct correlation between the metabolic activity of the gut microbiota and modifications in mycotoxin contents was not clearly observed, adsorptive phenomena to bacterial cells and to undigested food constituents might explain the observed modifications.

Highlights

Alternaria mycotoxins are low-molecular-weight compounds produced by different Alternaria species, among which Alternaria alternata is the most important mycotoxin-producing species of this genus of molds (EFSA 2011).Alternaria species can infect various fruits and vegetables and can produce, under favorable conditions of temperature and humidity, more than 70 structurally characterized toxins (Ostry 2008; EFSA 2011)
In contrast to what was observed for formamidopyrimidine DNA glycosylase (FPG)-untreated samples in which the DNA-strand-break ability of mycotoxins was almost completely quenched by the presence of the fecal material, the extent of FPG-sensitive sites was mitigated but not eliminated, with tail intensities ranging from 24.1 ± 4.70% (PM + CE) to 13.5 ± 2.47% (FS + CE)
Incubation with extract added to dead microorganisms (DM + CE) resulted in a lower level of FPG-sensitive sites compared to those recorded for samples containing living microorganisms, without reaching statistical significance after Bonferroni correction

Summary

Introduction

Alternaria mycotoxins are low-molecular-weight compounds produced by different Alternaria species, among which Alternaria alternata is the most important mycotoxin-producing species of this genus of molds (EFSA 2011).Alternaria species can infect various fruits and vegetables and can produce, under favorable conditions of temperature and humidity, more than 70 structurally characterized toxins (Ostry 2008; EFSA 2011). Among the substances which were most frequently reported in food and feed, both AOH and AME were found to act as topoisomerase poisons, leading to singleand double-DNA strand breaks, while ALT and iso-ALT did not show any genotoxic properties (Fehr et al 2009). The ability of AOH and AME to poison topoisomerases is thought to be a consequence of their planar structure, which would favor DNA pair-base intercalation (Dellafiora et al 2015). The perylene quinones ATX-II and STTX-III have been described to be even more genotoxic than AOH, acting probably through the formation of mycotoxin-DNA adducts as the main mechanism (Schwarz et al 2012b; Fleck et al 2016). As a matter of fact, both mycotoxins have been found to activate estrogen receptors (Lehmann et al 2006; Dellafiora et al 2018), and AOH can act as a full androgen agonist (Stypuła-Trębas et al 2017)

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