Role of topoisomerase inhibition and DNA repair mechanisms in the genotoxicity of alternariol and altertoxin-II

Abstract

Alternariol (AOH) and altertoxin-II (ALTX-II) have been demonstrated to possess genotoxic properties. However, the underlying mechanisms of action have not been fully elucidated yet. AOH has recently been shown to act as a topoisomerase I and II poison, contributing to its genotoxic properties. The topoisomerase-specific repair factor tyrosyl-DNA-phosphodiesterase-1 (TDP1) is involved in the respective repair processes of damaged DNA induced by topoisomerase II poison. In the present study, we investigated the role of DNA repair pathways for the extent of DNA damage by AOH and addressed the question whether interference with topoisomerase II might play a role in the genotoxicity of ALTX-II. Under cell-free conditions, AOH and ALTX-II suppressed the activity of topoisomerase II at a comparable concentration range. In HT29 cells, AOH enhanced the level of covalent DNA-topoisomerase II complexes, thus acting as a topoisomerase poison in DNA damaging concentrations. In contrast, ALTX-II in genotoxic concentrations did not show any effect on the stability of these complexes, indicating that interference with topoisomerases does not play a relevant role in genotoxicity. The differences in genotoxic mechanisms seem to be reflected in the activation of p53. AOH was found to increase p53 phosphorylation in HT29 cells in DNA damaging concentrations. In contrast, incubation with ALTX-II did not affect p53 phosphorylation despite substantial increase in tail intensity in the comet assay, suggesting that the DNA lesions formed by ALTX-II are not detected by the DNA-repair machinery of HT29 cells. These results are supported by differences in persistence of DNA damage, still maintained after 24 h for ALTX-II but nearly vanished already after 3 h for AOH. Furthermore, microarray and qPCR analysis did not indicate any substantial impact of AOH on the transcription of key elements of DNA repair pathways. However, siRNA-approaches indicate that, in addition to TDP1, the expression of other elements of the DNA repair machinery exemplified by the 70 kDa Ku autoantigen and the proliferating cell nuclear antigen are relevant for AOH-mediated DNA damage.

Activation of the Nrf2-ARE pathway by the Alternaria alternata mycotoxins altertoxin I and II

“…In fact, both molecules have been described as mutagenic in S. typhimurium strains TA98, TA100, TA1537, TA102 and TA104, yet ATX I was reported to be less potent than ATX II (Schrader et al 2001; Stack and Prival 1986). Similarly, this trend was confirmed for several other properties, such as: mutagenicity in V79 cells (Fleck et al 2012; Schrader et al 2006), ability to induce DNA strand breaks and formamidopyrimidine DNA glycosylase-sensitive sites in human cancer cell lines (Fleck et al 2014; Schwarz et al 2012b) or cytotoxicity in mammalian cell cultures (Boutin et al 1989; Tiessen et al 2013b). Although the toxicological impact of altertoxins has been investigated previously, studies dealing with the metabolic pathways of perylene quinone-type mycotoxins have been limited to the observations that ATX II can be reduced to ATX I by the colorectal adenocarcinoma cell line Caco2 and that ATX II can form adducts with GSH in a cell-free system (Fleck et al 2014).…”

“…This model was previously used as representative of intestinal cells and it is particularly relevant for the study of food contaminants (Schwarz et al 2012a; Tiessen et al 2013b). In control conditions, the fluorescent signal of Nrf2 (red) appears to be similar to that of Keap1 (green; Fig.…”

Dual effectiveness of Alternaria but not Fusarium mycotoxins against human topoisomerase II and bacterial gyrase

“…In previous studies, three secondary metabolites produced by food-contaminating fungi of the genus Alternaria, the dibenzo-α-pyrones AOH and AME, as well as the perylene-quinone ATX II, were found to inhibit the activity of human topo II in vitro (Fehr et al 2009; Tiessen et al 2013). Based on these results, the question arose what might prompt a mold to produce secondary metabolites that target human enzymes.…”

New perylenequinone derivatives from the endophytic fungus Alternaria tenuissima SS77

“…S1), belong to this second group of mold perylenequinones, 15 some of which have been reported as phytotoxins 16,17 and others have gained importance over the years for causing food and feed contaminations, offering risks to human health due to their mutagenic effects. [18][19][20][21] In plants, toxic effects of these perylenequinones may occur in the same way that of cercosporin, by generation of free radicals that damage host cells after photoactivation. 9 Mutagenicity observed in bacterial and mammal cells have been attributed to various mechanisms of action.…”

“…9 Mutagenicity observed in bacterial and mammal cells have been attributed to various mechanisms of action. 20,21 Particular structural features of different perylenequinone derivatives contribute to their photoactive and mutagenic effects, affording compounds with variable levels of bioactivity.…”