Toxic indole alkaloids avrainvillamide and stephacidin B produced by a biocide tolerant indoor mold Aspergillus westerdijkiae
Graphical abstract
Introduction
Potentially toxigenic primary storage molds, Aspergillus versicolor, Penicillium species, and Paecilomyces variotii are frequently reported connected to respiratory illness in hospitals (Lugauskas and Krisktaponis, 2004), private dwellings, schools (Pieckova and Kunova, 2002) and building materials (Andersen et al., 2011, Fisk et al., 2010, Mendell et al., 2011). These fungi are able to proliferate in materials with low water activity (aw < 0.8) and adapted to long periods of dryness (reviewed by Nielsen and Frisvad, 2011).
The large family of the bioactive indole alkaloids containing a bicyclo[2.2.2]diazaoctane core derived of tryptophan, proline and isoprene are produced by filamentous fungi Aspergillus and Penicillium (Ding et al., 2010, Finefield et al., 2012). Avrainvillamide (Fig. 1), its dimer stephacidin B (Fig. 1) and aspergamide A belong to the subfamily of these indole alkaloids where diketopiperazine group is an internal part of bicyclo[2.2.2]diazaoctane core (Fig. 1, highlighted by pale orange) with pyrano-indole and indole nitrone moieties (Baran et al., 2005). Other members, belonging to this subfamily but lacking the indole nitrone moiety, are stephacidin A (Qian-Cutrone et al., 2002), notoamides, versicolamide B and sclerotiamide (Miller et al., 2009, Tsukamoto et al., 2009, Kato et al., 2007). Avrainvillamide and stephacidin B, isolated from Aspergillus ochraceus WC7646, have antitumor properties and, hence, the reported cytotoxicity studies have been focused on human malignant cell lines (Qian-Cutrone et al., 2002, Wulff et al., 2007a, Wulff et al., 2007b, Fenical et al., 2000, Sugie et al., 2001).
In this paper we describe isolation of avrainvillamide and stephacidin B from Aspergillus westerdijkiae, and show its high toxicity toward nontumor mammalian cells is due to these toxins. We showed that the high toxicity was mediated by the nitrone group, present in avrainvillamide (Fig. 1) and in its dimer stephacidin B (Fig. 1) but absent in other members of the diketopiperazine group of indole alkaloids. We describe a potential mechanism for the nitrone-group mediated toxicity of the indole alkaloids avrainvillamide and stephacidin B, from A. westerdijkiae, and discuss the environmental determinants potentially promoting colonisation of indoor space by these Aspergilli posing a threat to human health.
Section snippets
Reagents and supplies
Valinomycin, citrinin, sterigmatocystin, emodin and ochratoxin A were from Sigma–Aldrich (St. Louis, MO, USA). Enniatin B was from Enzo Life Sciences AG (Lausanne, Switzerland), triclosan (2,4,4′-trichloro-2′-hydroxydiphenyl ether) from Merck (Darmstadt, Germany). Stephacidin A was obtained from AnalytiCon Discover (Potsdam, Germany). The fluorogenic dyes (JC-1, calcein AM (cell permeant, 1 mg mL−1 in DMSO), Hoechst 33342 (10 mg mL−1 in water) and propidium iodide (1.6 mg mL−1 in water) were
Detection of toxin producing Aspergillus westerdijkiae from buildings where the occupants suffered from building connected ill health
Fifty-six independent isolates from the dusts and on fall-out tryptic soy agar (TSA) plates from the troubled buildings were morphologically considered as Aspergillus spp and screened for toxicity using spermatozoan motility inhibition test. Six isolates (6/56, 10%) were found, of which the ethanolic exudate was at least ten-times more potency than extract similarly prepared from biomass of Trichoderma longibrachiatum DSM768 (Table 1). Three different toxicity endpoints (EC50) were recorded: 1.
Discussion
In this paper, we describe the indole alkaloids avrainvillamide and stephacidin B and their cytotoxicity to nontumor porcine cells. Avrainvillamide and stephacidin B were purified from A. westerdijkiae strains isolated from buildings troubled with adverse effects on human health connected to indoor air. The mass spectrometry analysis showed that the two toxic fractions found in cell extracts of several independent isolates of A. westerdijkiae strains had identical mass ions at m/z 891. Tandem
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This research was supported by Finnish Work Environment Fund (Grants 109124, 111084 and 112134), and the Academy of Finland (Grant 118637). The authors thank Päivi Pakalen and Risto Salin for innovative materials sampling and technical documentation. The Faculty Instrument Centre (Laitekeskus) for expert technical support and Tuula Suortti, Leena Steininger and Hannele Tukiainen for effective administration.
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These authors contributed equally to this work.