Total synthesis of dehydroaltenuene A. Revision of the structure and total synthesis of dihydroaltenuene B.

Total synthesis of alternaria toxins starting from previously synthesized altenuene (3) and isoaltenuene (4) is described. Dihydroaltenuene B (9) was prepared by hydrogenation of 3, and the non-natural epimer 3-epi-dihydroaltenuene A was obtained analogously from 4. Inspection of the spectroscopic data for 9 revealed that the originally proposed structure was in error. A revised structure (11), unambiguously proven by total synthesis, is reported herein. Oxidation of 4 with oxygen in the presence of palladium(II) acetate as catalyst led to the formation of dehydroaltenuene A (8), while oxidation of 3 using identical conditions yielded ent-dehydroaltenuene B (ent-9). Oxidation of 4 with manganese(IV) oxide furnished dehydroaltenusin (12), although only impure material was obtained in low yield.

Oxidative in vitro metabolism of the Alternaria toxins altenuene and isoaltenuene.

The mycotoxins altenuene (ALT) and isoaltenuene (iALT) frequently occur in food and feed items infested by fungi of the genus Alternaria, but nothing is known about their oxidative metabolism in mammals. We have therefore incubated ALT and iALT with microsomes from rat liver in the presence of a nicotinamide adenine dinucleotide phosphate (NADPH)-generating system and analyzed the extracted metabolites with HPLC and GC-MS after trimethylsilylation. Both toxins formed a major metabolite, which was tentatively identified as the 8-hydroxylation product by GC-MS analysis and by its methylation by the enzyme catechol-O-methyltransferase. Three minor metabolites were tentatively identified as 10-hydroxy- and two stereoisomers of 4-hydroxy-ALT and -iALT. The same metabolic pattern was observed in microsomes from different rat strains and from pigs and humans. Moreover, incubation of ALT with rat liver slices provided evidence that the same oxidative metabolites were formed under in vivo-like conditions. Thus, ALT and iALT exhibit a considerable propensity for undergoing metabolic hydroxylation reactions, and the toxicological properties of the oxidative metabolites should now be studied.

Alternariol acts as a topoisomerase poison, preferentially affecting the IIalpha isoform.

Alternariol (AOH), a mycotoxin formed by Alternaria alternata, has been reported to possess genotoxic properties. However, the underlying mechanism of action is unclear. Here, we tested the hypothesis that interactions with DNA-topoisomerases play a role in the DNA-damaging properties of AOH. First we compared DNA-damaging properties of AOH with other Alternaria mycotoxins such as AOH monomethyl ether (AME), altenuene and isoaltenuene. AOH and AME significantly increased the rate of DNA strand breaks in human carcinoma cells (HT29, A431) at micromolar concentrations, whereas altenuene and isoaltenuene did not affect DNA integrity up to 100 microM. Next, we selected AOH as the most DNA-damaging Alternaria metabolite for further studies of interactions with DNA topoisomerases. In cell-free assays, AOH potently inhibited DNA relaxation and stimulated DNA cleavage activities of topoisomerase I, IIalpha and IIbeta. Stabilisation of covalent topoisomerase II-DNA intermediates by AOH was also detectable in cell culture, and here, the IIalpha isoform was preferentially targeted. AOH is thus characterised as a poison of topoisomerase I and II with a certain selectivity for the IIalpha isoform. Since topoisomerase poisoning and DNA strand breakage occurred within the same concentration range, poisoning of topoisomerase I and II might at least contribute to the genotoxic properties of AOH.