Toxicity-guided fractionation was used to identify DNA strand breaking impact compounds in extracts obtained from rice heavily infested with the Alternaria alternata strains DSM 62006 and DSM 62010. The major genotoxic potential measured in the comet assay using human colon carcinoma cells (HT29) could be attributed to three unknown peaks, whereas the fractions containing alternariol, its monomethylether or tenuazonic acid showed no significant DNA damaging effects. According to (1)H and (13)C-NMR spectroscopy, one genotoxic impact compound was identified as Altertoxin II (ATXII). ATXII showed potent DNA damaging properties in HT29 cells with substantial induction of formamidopyrimidine DNA glycosylase (FPG)-sensitive sites. However, no effect was observed with respect to the cellular redox status, measured in the DCF assay and as total glutathione. The induction of apoptosis could be excluded as a potential reason for enhanced DNA damage. After 24 h of incubation with 1 μM ATX II, a significant increase of cells in the G(0)/G(1) phase was observed together with an inhibition of cell proliferation in the sulforhodamine B assay. Taken together, ATX II was found to contribute substantially to the genotoxic effects of complex extracts obtained from Alternaria alternata infested rice. The results demonstrate the high genotoxic potency of ATX II in human cells, underlining the necessity for further studies on the occurrence in food and its relevance for food safety.
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.
Studies on the genotoxicity of Alternaria mycotoxins focus primarily on the native compounds. Alternariol (AOH) and its methyl ether (AME) have been reported to represent substrates for cytochrome P450 enzymes, generating hydroxylated metabolites. The impact of these phase I metabolites on genotoxicity remains unknown. In the present study, the synthesis and the toxicological effects of the metabolites 4-hydroxy alternariol (4-OH-AOH) and 4-hydroxy alternariol monomethyl ether (4-OH-AME) are presented and compared to the effects of the parent molecules. Although the two phase I metabolites contain a catecholic structure, which is expected to be involved in redox cycling, only 4-OH-AOH increased reactive oxygen species (ROS) in human esophageal cells (KYSE510), 4 times more pronounced than AOH. No ROS induction was observed for 4-OH-AME, although the parent compound showed some minor impact. Under cell-free conditions, both metabolites inhibited topoisomerase II activity comparable to their parent compounds. In KYSE510 cells, both metabolites were found to enhance the level of transient DNA–topoisomerase complexes in the ICE assay. Although the level of ROS was significantly increased by 4-OH-AOH, neither DNA strand breaks nor enhanced levels of formamidopyrimidine-DNA-glycosylase (FPG)-sensitive sites were observed. In contrast, AOH induced significant DNA damage in KYSE510 cells. Less pronounced or even absent effects of hydroxylated metabolites compared to the parent compounds might at least partly be explained by their poor cellular uptake. Glucuronidation as well as sulfation appear to have only a minor influence. Instead, methylation of 4-OH-AOH seems to be the preferred way of metabolism in KYSE510 cells, whereby the toxicological relevance of the methylation product remains to be clarified.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-016-1801-0) contains supplementary material, which is available to authorized users.
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