A toxicological evaluation of the interactions between alternaria mycotoxins, food constituents and gut microbiota: implications for human health

Abstract

Alternaria molds can produce more than seventy secondary toxic metabolites, some of which might pose a severe threat to human health due to both the adverse effects observed in vitro and in vivo (genotoxic, mutagenic, clastogenic, androgenic, estrogenic, fetotoxic and teratogenic effects) and the frequent occurrence in food. Despite this, the shortage of toxicological and occurrence data on this class of food contaminants hinders the proper assessment of risks to human health and, consequently, it prevents the establishment of maximum levels in food. In this context, the risk assessment on chemical contaminants is currently based on the integration of knowledges about the occurrence and toxicity of a given compound, without taking into account that food is often contaminated by multiple xenobiotics and that co-exposure to multiple chemicals can result in effects different from those exerted by the mixture components taken individually (i.e. additive, synergistic or antagonistic effects). Since the latest call for data on Alternaria mycotoxins inquired by EFSA in 2016, many steps have been taken to increase knowledge on this class of mycotoxins. However, many efforts are still needed to clarify the actual co-occurrence of multiple Alternaria mycotoxins in food, as well as the effects that Alternaria mycotoxin mixtures may exert. In addition, few data are currently available about the possible interaction of Alternaria mycotoxins with gut microbiota, microbial metabolites and food constituents, which might modify the toxicokinetic and toxicodynamic of these fungal metabolites, thus potentially resulting in the modification of the final toxicological outcome. On the other hand, the capability of Alternaria mycotoxins to mediate indirect negative effects on human health by targeting the gut microbiota has rarely been assessed. Considering that modifications of the composition and/or activity of the gut microbiota might influence both normal physiology and disease susceptibility of the host, the effects of Alternaria mycotoxins on the latter should also be investigated. Based on this, the aims of the present PhD thesis were to: i) summarize the most relevant literature concerning the occurrence and toxicity of Alternaria mycotoxins, studied either individually or in combination with other mycotoxins or bioactive compounds of food origin; ii) investigate the ability of gut microbiota, microbial metabolites and food constituents to modify the bioavailability and/or bioactivity of Alternaria mycotoxins; iii) evaluate the capability of Alternaria mycotoxins to affect the growth and/or the activity (biofilm production) of human gut bacterial strains. The main results obtained during the PhD program are reported below. a) According to the literature reviewed, contaminated foods often contain more than one Alternaria mycotoxin and, in some cases, these fungal metabolites are present in food along with mycotoxins produced by other genera of molds, such as those of Fusarium, Penicillium and Aspergillus. These mycotoxins can be found not only in fresh and processed foods, but also in food supplements, in which a high number of mycotoxins may be present. As for the effects exerted by mycotoxin mixtures, several studies reported the onset of additive, synergistic or antagonistic effects following treatments with multiple Alternaria mycotoxins, as well as Alternaria mycotoxins in combination with other types of mycotoxins or food bioactives. b) Undigested food constituents and gut microorganisms from human feces were found to modify, probably through adsorptive phenomena, the concentrations of mycotoxins contained in a complex extract of cultured Alternaria fungi. These modifications resulted in a reduced ability of the Alternaria extract to induce DNA strand breaks in the HT-29 cell line. The suppression of the direct genotoxic effects was mainly related to the loss of the epoxide-carrying Alternaria mycotoxins altertoxin-I and stemphyltoxin-III. Contrary to what was observed for DNA strand breaks, the ability of the Alternaria extract to induce formamidopyrimidine-DNA glycosylase-sensitive sites was only slightly affected by the contact with the various feces-derived fractions and was still present after 3h of anaerobic incubation. c) Despite the contact of the Alternaria extract with the fecal materials resulted in complete or partial losses of the mycotoxins, the extract components were found to elicit antiestrogenic effects in the Ishikawa cell line even after 3h of anaerobic incubation with the fecal materials. In particular, the intrinsic estrogenic properties of fecal slurries were antagonized by the extract components probably due to the ability of the latter to reduce the ER-α/ER-β nuclear ratio, while a possible action of the mycotoxins as ER-antagonists was excluded through an in silico study. d) Human gut bacterial strains belonging to five of the most dominant gut microbial phyla were found to adsorb the Alternaria mycotoxins alternariol, alternariol monomethyl ether, altertoxin-I, alterperylenol, and altersetin, thus reducing the free-absorbable portion of mycotoxins. Of note, the tendency of mycotoxins to accumulate within bacteria, especially in Gram-negative strains, was found to be directly related to their lipophilicity values (i.e. greater accumulation for mycotoxins having higher lipophilicity values). On the other hand, the mycotoxins of the Alternaria extract were found to affect the growth of most of the strains tested and to suppress the formation of biofilms. e) The gut microbiota might exert indirect protective effects on human health by producing compounds able to compete with the mycotoxins for the binding to cellular proteins such as transporters and enzymes. In particular, the gut microbial metabolite urolithin C (which is structurally similar to the Alternaria mycotoxin alternariol) was found to reduce the absorption of alternariol in a Caco-2 cell model. In addition, this microbial metabolite also impaired the phase II metabolism of the mycotoxin, which resulted mainly in a reduced production of alternariol-sulphates. From a mechanistic point of view, the suppression of sulphates production was probably a consequence of the ability of urolithin C to competitively bind to sulfotransferases with a theoretically higher affinity compared to the mycotoxin. In conclusion, results obtained during these three-years PhD program highlight the need to evaluate the effects exerted by Alternaria mycotoxins in complex mixtures rather than singularly, given the high frequency of co-occurrence of multiple mycotoxins in food and the possible combined effects that could derive from exposure to chemical mixtures. In addition, the experimental works included in this PhD thesis demonstrate that interactions of Alternaria mycotoxins with food constituents, natural or microbial metabolites, as well as with gut microorganisms might occur after ingestion of contaminated foods, thus resulting in modifications of the bioavailability or bioactivity of mycotoxins. Finally, the potential ability of Alternaria mycotoxins to affect in vitro the growth and activity of human gut bacterial strains was also shown. In this light, considering the known limitations deriving from in vitro studies and the importance of the gut microbiota for human health, the future in vivo investigation of the overall effects of the Alternaria mycotoxins on the gut microbiota would be desirable in order to pave the way for an all-round assessment of the actual risk related to this class of food contaminants.