Impact of phase I metabolism on uptake, oxidative stress and genotoxicity of the emerging mycotoxin alternariol and its monomethyl ether in esophageal cells.

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.

Modulation of the cellular redox status by the Alternaria toxins alternariol and alternariol monomethyl ether.

The mycotoxin alternariol (AOH) has been reported to possess genotoxic properties, inducing enhanced levels of DNA damage after only 1 h of incubation. In the present study we addressed the question whether the induction of oxidative stress might contribute to the genotoxic effects of AOH or its naturally occurring monomethylether (AME). In the dichlorofluorescein (DCF) assay, treatment of HT29 cells for 1 h enhanced the formation of dichlorofluorescein, indicative for ROS formation. The total glutathione (tGSH) was transiently decreased. In accordance with the results of the DCF assay, AOH and AME enhanced the proportion of the transcription factor Nrf2 in the nucleus. Concomitantly, the Nrf2/ARE-dependent genes γ-glutamylcysteine ligase (γ-GCL) and glutathione-S-transferase (GSTA1/2) showed enhanced transcript levels. After 24 h of incubation this effect was also reflected on the protein level by an increase of GST activity. However, in spite of the positive DCF assay and the activation of the redox-sensitive Nrf2/ARE-pathway, the level of oxidative DNA damage, measured in the comet assay by the addition of formamidopyrimidine-DNA-glycosylase (fpg) remained unaffected. Of note, after 3 h of incubation no significant DNA damaging potential of AOH and AME was detectable, indicating either inactivation of the compounds or enhanced DNA repair. In summary, the mycotoxins AOH and AME were found to modulate the redox balance of HT29 cells but without apparent negative effect on DNA integrity.

Characterization of a genotoxic impact compound in Alternaria alternata infested rice as Altertoxin II.

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.