Polymethoxy-1-alkenes from Aphanizomenon ovalisporum inhibit vertebrate development in the zebrafish (Danio rerio) embryo model

Abstract

Cyanobacteria are recognized producers of a wide array of toxic or otherwise bioactive secondary metabolites. The present study utilized the zebrafish (Danio rerio) embryo as an aquatic animal model of vertebrate development to identify, purify and characterize lipophilic inhibitors of development (i.e., developmental toxins) from an isolate of the freshwater cyanobacterial species, Aphanizomenon ovalisporum.Bioassay-guided fractionation led to the purification, and subsequent chemical characterization, of an apparent homologous series of isotactic polymethoxy-1-alkenes (1-6), including three congeners (4-6) previously identified from the strain, and two variants previously identified from other species (2 and 3), as well as one apparently novel member of the series (1). Five of the PMAs in the series (1-5) were purified in sufficient quantity for comparative toxicological characterization, and toxicity in the zebrafish embryo model was found to generally correlate with relative chain length and/or methoxylation. Moreover, exposure of embryos to a combination of variants indicates an apparent synergistic interaction between the congeners. Although PMAs have been identified previously in cyanobacteria, this is the first report of their apparent toxicity. These results, along with the previously reported presence of the PMAs from several cyanobacterial species, suggest a possibly widespread distribution of the PMAs as toxic secondary metabolites and warrants further chemical and toxicological investigation.

Keywords: Aphanizomenon ovalisporum; cyanobacteria; harmful algal blooms (HABs); polymethoxy-1-alkenes; toxins; vertebrate development; zebrafish (Danio rerio) embryo.

Figure 1

Structure of polymethoxy Alkenes (1–6) Isolated in the Current and Previous [13,14,15,16] Studies.

Figure 2

Chromatogram Showing PMAs (1–6) Isolated by HPLC. See Experimental Section for details.

Figure 3

Developmental Toxicity of PMAs (1–4) Isolated from A. ovalisporum (Lake Kinneret, Israel). Photomicrographs of embryos at 4 dpf. Shown are embryos exposed to 1–4 at 50 µg mL⁻¹ (A–D, respectively) and 100 µg mL⁻¹ (E–H, respectively). Untreated control embryo at 4 dpf (I) shown for comparison. Arrows indicate areas where blood is pooled adjacent to the heart. Images in A–C and E–G, are not to scale with D, H and I.

Figure 4

Apparent Synergistic Interactions in the Developmental Toxicity of 3 and 4. Shown are embryos exposed to 50 µg mL⁻¹ of 3 (A), and 100 µg mL⁻¹ of 3 (B) and 4 (C), compared to an equivalent combined total concentration (i.e., 25 and 50 µg mL⁻¹ of each) of the two variants (D and E, respectively). Embryos shown at 4 dpf.