Elsevier

Toxicon

Volume 99, 1 June 2015, Pages 58-67
Toxicon

Toxic indole alkaloids avrainvillamide and stephacidin B produced by a biocide tolerant indoor mold Aspergillus westerdijkiae

https://doi.org/10.1016/j.toxicon.2015.03.011Get rights and content

Highlights

  • Aspergillus westerdijkiae from sick buildings emit toxic nitrone indole alkaloids.

  • Nitrone groups potentiate 100-fold the toxicity of indole alkaloid avrainvillamide.

  • 2–7 % of indoor Aspergillus lipids was avrainvillamide or stephacidin B.

  • Boron and guanide biocides enhances sporulation of toxigenic Aspergillus.

  • Biocides cause toxicological risks giving benefit to the toxic fungi such as A. westerdijkiae.

Abstract

Toxic Aspergillus westerdijkiae were present in house dust and indoor air fall-out from a residence and a kindergarten where the occupants suffered from building related ill health. The A. westerdijkiae isolates produced indole alkaloids avrainvillamide (445 Da) and its dimer stephacidin B (890 Da). It grew and sporulated in presence of high concentrations of boron or polyguanidine (PHMB, PHMG) based antimicrobial biocides used to remediate mold infested buildings. The boar sperm cells were used as sensor cells to purify toxins from HPLC fractions of the fungal biomass. Submicromolar concentrations (EC50 0.3–0.4 μM) blocked boar spermatozoan motility and killed porcine kidney tubular epithelial cells (PK-15). Plate grown hyphal mass of the A. westerdijkiae isolates contained 300–750 ng of avrainvillamide and 30–300 ng of stephacidin B per mg (wet weight). The toxins induced rapid (30 min) loss of boar sperm motility, followed (24 h) by loss of mitochondrial membrane potential (ΔΨm). Apoptotic cell death was observed in PK-15 cell monolayers, prior to cessation of glucose uptake or loss of ΔΨm. Avrainvillamide and stephacidin B were 100-fold more potent towards the porcine cells than the mycotoxins stephacidin A, ochratoxin A, sterigmatocystin and citrinin. The high toxicity of stephacidin B indicates a role of nitrone group in the mechanism of toxicity. Avrainvillamide and stephacidin B represent a new class of toxins with possible a threat to human health in buildings. Furthermore, the use of biocides highly enhanced the growth of toxigenic A. westerdijkiae.

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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