On the Hunt for New Toxin Families Produced by a Mediterranean Strain of the Benthic Dinoflagellate Ostreopsis cf. ovata

Abstract

Ostreopsis cf. ovata is a benthic dinoflagellate known to produce palytoxin (PLTX) and its analogues. Recent investigations suggested the production of unknown toxins by a Mediterranean strain. In the present work, two new families of toxins, potentially novel in their structures, were purified from this same Mediterranean strain of Ostreopsis cf. ovata. The low amount of material isolated only allowed for acquisition of high-resolution mass spectrometry data and the evaluation of their cytotoxicity to human lung cancer cells. Based on their HRMS data, none of these new compounds appear to be close PLTX analogues, although their mass spectra suggest poly-hydroxylated long chain compounds of high molecular weight (1370-2143 Da). The cell cytotoxicity concentrations (CC₅₀) of these new purified toxins ranged between 0.68 and 3.12 µg/mL, and this was enhanced when they were tested as mixtures, suggesting synergistic effects of Ostreopsis toxins. The two families of compounds were named the liguriatoxins (LGTX) and rivieratoxins (RVTX), with each family containing three members. Additional work on purification is needed to fully characterize the structures of these six new dinoflagellate toxins.

Keywords: Ostreopsis; cytotoxicity; high resolution mass spectra; polyhydroxy compounds; toxins.

Figure 1

Percent survival of the NCI-H460 cell line as a result of treatment with O. cf. ovata fractions: (A) the aqueous (OST-AQ) and organic (OST-OR) extracts at 1 and 10 µg/mL (B) 1 and 10 µg/mL of the fractions OF1 through OF12, and (C) 5, 2, 0.5, 0.05, 0.005, and 0.0005 µg/mL of the fractions OF5 through OF10. A concentration of 1% DMSO in RPMI 1640 was chosen to serve as a negative control (set to 100% survival) in all calculations to establish consistency by matching the media DMSO composition of all the samples, while 2 µg/mL doxorubicin in RPMI 1640 served as a positive control. Statistical significance values were calculated using 2-way ANOVA function for panels (A) (p < 0.0001 ****) and (B,C) (adjusted p = 0.0001 *** and p < 0.05 *) (in GraphPad 9.3.1 GraphPad Software Inc., San Diego, CA, USA). No statistical significance was signified using ns.

Figure 1

Percent survival of the NCI-H460 cell line as a result of treatment with O. cf. ovata fractions: (A) the aqueous (OST-AQ) and organic (OST-OR) extracts at 1 and 10 µg/mL (B) 1 and 10 µg/mL of the fractions OF1 through OF12, and (C) 5, 2, 0.5, 0.05, 0.005, and 0.0005 µg/mL of the fractions OF5 through OF10. A concentration of 1% DMSO in RPMI 1640 was chosen to serve as a negative control (set to 100% survival) in all calculations to establish consistency by matching the media DMSO composition of all the samples, while 2 µg/mL doxorubicin in RPMI 1640 served as a positive control. Statistical significance values were calculated using 2-way ANOVA function for panels (A) (p < 0.0001 ****) and (B,C) (adjusted p = 0.0001 *** and p < 0.05 *) (in GraphPad 9.3.1 GraphPad Software Inc., San Diego, CA, USA). No statistical significance was signified using ns.

Figure 2

Mass spectra of fractions OF6-2 and -3 (LGTX A) obtained from (A) a full scan acquisition, showing the monoisotopic ion and the multi-charged ions (upper panel), as well as fragmentation experiments on (B) the monoisotopic ion (lower left panel), and (C) the [M+H+K]²⁺ m/z 1007.0236 (lower right panel).

Figure 2

Mass spectra of fractions OF6-2 and -3 (LGTX A) obtained from (A) a full scan acquisition, showing the monoisotopic ion and the multi-charged ions (upper panel), as well as fragmentation experiments on (B) the monoisotopic ion (lower left panel), and (C) the [M+H+K]²⁺ m/z 1007.0236 (lower right panel).

Figure 3

Mass spectra of OF6-6 obtained for (A) a full scan acquisition, showing the monoisotopic and the and multi-charged ions of LGTX B and C (upper panel), as well as fragmentation experiments on the (B) monoisotopic ion of LGTX B (lower left panel) and (C) the [M+2H]²⁺ ion m/z 1072.0603 of LGTX C (lower right panel).

Figure 3

Mass spectra of OF6-6 obtained for (A) a full scan acquisition, showing the monoisotopic and the and multi-charged ions of LGTX B and C (upper panel), as well as fragmentation experiments on the (B) monoisotopic ion of LGTX B (lower left panel) and (C) the [M+2H]²⁺ ion m/z 1072.0603 of LGTX C (lower right panel).

Figure 4

Mass spectra for fractions OF6-12, OF6-15, and OF6-18 (RVTX A, B and C) obtained from (A) a full scan acquisition (upper panel) and (B) fragmentation of the [M+H+Na]²⁺ ion for each fraction (lower panel).

Figure 4

Mass spectra for fractions OF6-12, OF6-15, and OF6-18 (RVTX A, B and C) obtained from (A) a full scan acquisition (upper panel) and (B) fragmentation of the [M+H+Na]²⁺ ion for each fraction (lower panel).

Figure 5

GNPS molecular network obtained from HRMS/MS data of all fractions OF6-2 to OF6-18. All blue nodes were ions detected in at least one fraction, while the orange node corresponds to an ion detected in the library of cyanobacterial natural products. Single nodes mark the absence of common MS² fragments with those of any other compound. The node size corresponds to the precursor ion intensity. The absence of clear fragmentations for LGTX B (only a suite of water losses was observed) precluded its incorporation in the network, whereas LGTX A and C, as well as the RVTX A, B, and C, were all incorporated. Cosine scores on the edges indicate the similarity between two nodes based on their MS² fragments.