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

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.

Keywords: Bacteria; Chemical mixture; Food contaminant; Genotoxicity; Microbiome; Mold.

Fig. 1

DNA strand breaks (-FPG) and additional FPG-sensitive lesions (+ FPG) measured with the comet assay in HT-29 cells after 1 h treatment with the samples collected before (0 h) and after (3 h) anaerobic incubations with fecal fractions. Cells were exposed to samples diluted 1:10 with DMEM to reach an extract concentration of 5 µg/mL. All values are expressed as mean + SD of four independent biological experiments. Significant differences between the extract in PBS (“PBS + CE”) and the other samples were calculated by one-way ANOVA, followed by Bonferroni post hoc test (p < 0.01), with “a”, “b”, “c”, and “d”, indicating a significant difference to the FPG-untreated PBS + CE samples before incubation, to the FPG-treated PBS + CE samples before incubation, to the FPG-untreated PBS + CE samples after incubation, and to the FPG-treated PBS + CE samples after incubation, respectively. Differences between tail intensity values found before and after anaerobic incubation for each sample were calculated by Student’s t test (*p < 0.05; **p < 0.01). Negative 0.1% DMSO, Positive UV light treatment, PBS + CE extract dissolved in PBS, FS + DMSO fecal slurry + DMSO, FW + DMSO filtered fecal water + DMSO, FS + CE fecal slurry + extract, FW + CE filtered fecal water + extract, PM + CE fecal particulate matter + extract, LM + CE living microorganisms + extract, DM + CE dead microorganisms + extract

Fig. 2

Bar charts highlighting mycotoxins affected by contact with fecal material. All values are expressed as mean + SD of four independent biological experiments. Differences between concentrations found before and after anaerobic incubation for each sample were calculated by Student’s t test (*p < 0.05; **p < 0.01). Significant differences between mycotoxin concentrations found in the extract-containing PBS control (“PBS + CE”) and the other samples were calculated by one-way ANOVA, followed by Bonferroni post hoc test (p < 0.01). a Significant difference between the mycotoxin concentration found in samples containing fecal material before incubation and the PBS + CE control before incubation; b significant difference between the mycotoxin concentration found in samples containing fecal material after incubation and the PBS + CE control after incubation. Letters followed by “#” indicate a significant difference at p < 0.05. PBS + CE extract dissolved in PBS, FS + CE fecal slurry + extract, FW + CE filtered fecal water + extract, PM + CE fecal particulate matter + extract, LM + CE living microorganisms + extract, DM + CE dead microorganisms + extract, a.u.c. area under the curve