# Toxic responses of metabolites produced by Ostreopsis cf. ovata on a panel of cell types

Rachelle Lanceleur, Marin-Pierre Gemin, Anne Louise Blier, Lisa Meslier, Damien Reveillon, Zouher Amzil, Eva Ternon, Olivier P. Thomas, Valérie Fessard

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## To cite this version:

Rachelle Lanceleur, Marin-Pierre Gemin, Anne Louise Blier, Lisa Meslier, Damien Reveillon, et al. Toxic responses of metabolites produced by Ostreopsis cf. ovata on a panel of cell types. Toxicon, 2024, 240, 107631 (9p.). 10.1016/j.toxicon.2024.107631. hal-04573218

HAL Id: hal-04573218

https://hal.science/hal-04573218v1

Submitted on 15 Sep 2025

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Distributed under a Creative Commons Attribution 4.0 International License
# Toxic responses of metabolites produced by *Ostreopsis cf. ovata* on a panel of cell types

Lanceleur Rachelle <fnref n="1" />, Gemin Marin-Pierre <fnref n="2" />, Blier Anne Louise <fnref n="1" />, Meslier Lisa <fnref n="1" />, Reveillon Damien <fnref n="2" />, Amzil Zouher <fnref n="2" />, Ternon Eva <fnref n="3" />, <fnref n="4" />, Thomas Olivier P. <fnref n="5" />, Fessard Valérie <fnref n="1" />, *

## Abstract :

Blooms of the dinoflagellate *Ostreopsis cf. ovata* are regularly associated with human intoxications that are attributed to ovatoxins (OVTXs), the main toxic compounds produced by this organism and close analogs to palytoxin (PITX). Unlike for PITX, information on OVTXs'toxicity are scarce due to the absence of commercial standards. Extracts from two cultures of Mediterranean strains of O. cf. ovata (MCCV54 and MCCV55), two fractions containing or not OVTXs (prepared from the MCCV54 extract) and OVTX-a and -d (isolated from the MCCV55 extract) were generated. These chemical samples and PITX were tested on a panel of cell types from several organs and tissues (skin, intestine, lung, liver and nervous system). The MCCV55 extract, containing a 2-fold higher amount of OVTXs than MCCV54 extract, was shown to be more cytotoxic on all the cell lines and more prone to increase interleukin-8 (IL-8) release in keratinocytes. The fraction containing OVTXs was also cytotoxic on the cell lines tested but induced IL-8 release only in liver cells. Unexpectedly, the cell lines tested showed the same sensitivity to the fraction that does not contain OVTXs. With this fraction, a pro-inflammatory effect was shown both in lung and liver cells. The level of cytotoxicity was similar for OVTX-a and -d, except on intestinal and skin cells where a weak difference of toxicity was observed. Among the 3 toxins, only PITX induced a pro-inflammatory effect mostly on keratinocytes. These results suggest that the ubiquitous Na+/K+ ATPase target of PITX is likely shared with OVTX-a and -d, although the differences in pro-inflammatory effect must be explained by other mechanisms.

<footnote n="1">¹ ANSES, Fougères Laboratory, Toxicology of Contaminants Unit, French Agency for Food, Environmental and Occupational Health & Safety, Fougères, 35 306, France</footnote>

<footnote n="2">² IFREMER, PHYTOX, METALG Laboratory, 44000, Nantes, France</footnote>

<footnote n="3">³ Sorbonne Université, CNRS, Laboratoire d'Océanographie de Villefranche, UMR 7093, BP 28, 06230, Villefranche-sur-Mer, France</footnote>

<footnote n="4">⁴ Université Côte d'Azur, CNRS, OCA, IRD, Géoazur, 250 rue Albert Einstein, 06560, Valbonne, France</footnote>

<footnote n="5">⁵ School of Biological and Chemical Sciences and Ryan Institute, University of Galway, University Road, H91TK33, Galway, Ireland</footnote>

<footnote>* Corresponding author : Valérie Fessard, email address : valerie.fessard@anses.fr</footnote>
**Graphical abstract**

<figure><img src="image_3.png" /></figure>

**Highlights**

► Extracts of *Ostreopsis* cf. *ovata* were cytotoxic to a broad range of cell lines. ► The fraction not containing ovatoxins induced IL-8 release on liver and lung cells. ► PITX was more cytotoxic than OVTX-a and –d on cells from intestine and the nervous system. ► A similar IC₅₀ was observed on lung cells for the three toxins. ► OVTX-a and –d did not induce IL-8 release on intestinal, lung and skin cells.

**Keywords**: ovatoxins, palytoxin, in vitro, pro-inflammatory effect, cytotoxicity
44 Introduction

Human intoxications through aerosol inhalation as well as dermal and ocular contacts are recorded regularly along the coasts of the Northwestern Mediterranean Sea including Italy, France and Spain where blooms of the dinoflagellate *Ostreopsis* cf. *ovata* occur (Berdal et al. 2022). Recently, similar intoxications have been reported in the Bay of Biscay (Atlantic Ocean) (Paradis and Labadie 2022), together with the presence of *O. cf. ovata* and *O. cf. siamensis* in the same areas (Chomérat et al. 2022). Although the toxic compounds responsible for such effects have not been clearly identified (Berdal 2020), *O. cf. ovata* was shown to produce palytoxin (PITX)-like compounds named ovatoxins (OVTXs) (Ciminiello et al. 2012). Several OVTX analogs (a to l) have been identified in different species of the genus *Ostreopsis* (Patocka et al. 2018) but their purification from dinoflagellate cultures has always been described as challenging, limiting the toxicological studies required to carry out hazard assessment of OVTXs. As a consequence, only few results with pure OVTX-a have been published so far, while PITX toxicity has been more largely investigated (Patocka et al. 2015).

PITX has been shown to inhibit the Na+/K+ ATPase (Habermann 1989), located in the plasma membrane of animal cells (Mobasheri et al. 2000). It has also been suggested that the H+/Na+ ATPase may be a PITX target (Scheiner-Bobis et al. 2002; Patocka et al. 2018). Moreover, PITX disrupts the regulation of signaling pathways through different mechanisms depending on the cell type, as described for the MAP kinases activity (Wattenberg 2011). Consequently, PITX has been shown to affect a broad range of cells including intestinal cells (Valverde et al. 2008; Pelin et al. 2012; Fernández et al. 2013), neuronal cells (Vale et al. 2007; Louzao et al. 2008; Nicolas et al. 2015), erythrocytes, macrophages and splenocytes (Satoh et al. 2003; Aiba et al. 2005; Crinelli et al. 2012; Pezzolesi et al. 2012), aortic smooth muscle cells (Sheridan et al. 2005), skin cells (Pelin et al. 2013 ; Pelin et al. 2016a) and carcinoma cells (Bellocci et al. 2008; Görögh et al. 2013). Studies are now needed to compare the cell toxicity of OVTXs with PITX. Up to now, inconsistent results on the *in vitro* toxicity of OVTX-a compared to PITX have been reported. Indeed, while OVTX-a was first shown to be far less potent than PITX in inducing cytotoxicity on HaCaT keratinocytes (100 fold) and in the hemolytic test (10 fold) (Pelin et al. 2016b), similar activities were measured with PITX, OVTX-a or extracts containing OVTXs in the hemolytic test (Pezzolesi et al. 2012; Poli et al. 2018), and in the *Artemia franciscana* bioassay (Pavaux et al. 2020). Importantly, *in vivo* toxicity assessments in rodents after inhalation exposure of OVTX-a and PITX have
resulted in close values for lethality (50% lethal dose, LD₅₀) as well as similar symptoms and toxic effects (Poli et al. 2018).

In this study, the toxicity of extracts, fractions and pure OVTX-a and -d obtained from cultures of Mediterranean strains of O. cf. ovata were investigated on a panel of cell types, representative of several organs and tissues: skin, intestine, liver, lung and nervous system. The mitochondrial activity was measured through the MTT assay and the pro-inflammatory effect was detected by the release of interleukin 8 (IL-8). Finally, the results obtained with OVTX-a and -d were compared with the data obtained with PITX.

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# Material and methods

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## Chemicals

Cell culture products were purchased from Gibco (Cergy-Pontoise, France). Bovine serum albumin (BSA), 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), streptavidin horseradish peroxide (HRP) were supplied by Sigma-Aldrich (Saint Quentin Fallavier, France). Primary IL-8 antibody (M801), biotin-conjugated human IL-8 (M802B), recombinant IL-8 cytokine, SuperBlock blocking buffer, 3,3',5,5'-tetramethylbenzidine (TMB), Tween 20, Tumor Necrosis Factor alpha (TNFα) and sulfuric acid were obtained from Thermofisher Scientific. Monoclonal IL-8, biotinylated monoclonal IL-8 antibodies, SuperBlock blocking buffer, TNFα and TMB were purchased from ThermoFisher Scientific (Waltham, MA). Palytoxin (purity = 85.8%) was purchased from Wako (Pure Chemical Industries, Ltd., Japan) and solubilized in dimethylsulfoxide (DMSO).

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## Extracts and fractions from O. cf. ovata and purified OVTX-a and -d

The same extracts prepared from cultures of O. cf. ovata (MCCV54 and MCCV55) as well as the same purified OVTX-a and -d used in Gémin *et al.* (2022) were tested in the current study. Two fractions prepared from the extract MCCV54 were also tested. They were obtained as described previously: Pavaux *et al.* (2020) for fractions of O. cf. ovata containing or not OVTXs (named OVTX+ and OVTX- respectively) and Gémin *et al.* (2022) for extracts of the strains MCCV54 and MCCV55, and pure OVTX-a and -d. Briefly, two strains of O. cf. ovata (MCCV54 and MCCV55) were obtained from the Mediterranean Culture Collection of Villefranche-sur-Mer (France). Cells were cultivated and harvested as described in Gémin *et al.* (2022) to collect 75 g of MCCV54 and 35 g of MCCV55 (wet pellets). Extracts of both strains were prepared by adding MeOH/H₂O (1:1 v/v) on 5 g cell pellets (ratio: 4 mL/g of wet pellet) and the suspension was vortexed and centrifuged at 4000 g during 5 min. The supernatant of each extraction process was concentrated by evaporation under a nitrogen flow (N-evap, Organomation) at 30 °C. Two fractions were then prepared from MCCV54 following size exclusion chromatography: one combining fractions where OVTX-a to -f were detected (OVTX+) by MS; one combining fractions where those metabolites were not detected (OVTX-) (see Pavaux *et al.* (2020) for details). A similar fraction OVTX+ prepared from the MCCV55 extract was further purified by semi-preparative chromatography to obtain 223 and 10.5 µg of OVTX-a and -d respectively (see Gémin *et al.* (2022) for details). The fraction OVTX- was dried by rotary evaporation (Büchi rotavapor R-200), weighted and then dissolved into ultra-pure water (at 50 mg/mL). The fraction OVTX+ was not evaporated completely and therefore could not be expressed in ng/mL. However, the different OVTXs were quantified (see below) and the total quantity of OVTXs present in the fraction was estimated in
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PITX equivalent, considering an equivalent factor of 1 for each variant. Extracts from MCCV54 and MCCV55, OVTX+ fraction of MCCV54 as well as the purified OVTX-a and -d were concentrated in DMSO prior to toxicity investigations. The quantification of OVTXs was performed by LC-MS/MS as described in Gémin *et al.* (2022).

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Cell cultures

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Human intestinal Caco2 cells (HTB-37), obtained from the American Type Culture Collection (ATCC) (Manasas, VA), were maintained in Minimum Essential Medium containing 5.5 mM D-glucose, Earle's salts and 2 mM L-alanyl-glutamine (MEM GlutaMAX™) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids, 50 IU/mL penicillin and 50 µg/mL streptomycin. Non-differentiated cells were used at passages 29 to 42.

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Rat enteric glial cell line (EGC) (CRL2690) was obtained from the American Type Culture Collection (ATCC, Manasas, VA). Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 25 mM D-Glucose and 4 mM L-glutamine, supplemented with 10% heat-inactivated fetal calf serum (FCS), 50 IU/mL penicillin and 50 µg/mL streptomycin. Cells were used at passages 45 to 65.

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Human lung A549 were obtained from the American Type Culture Collection (CRM-CCL-185), and were cultured in DMEM containing 5.5 mM D-glucose and 4 mM L-glutamine supplemented with 10% heat-inactivated fetal calf serum (FCS), 50 U/mL penicillin, and 50 µg/mL streptomycin. Cells were used at passages 10 to 30.

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Human liver HepaRG cells were purchased from Biopredic international (Rennes, France) and were cultured in Williams E medium supplemented with 10% FCS, 50 U/mL penicillin, 50 µg/mL streptomycin, 5 µg/mL insulin, 2 mM glutamine and 50 µM hydrocortisone hemisuccinate. Non-differentiated cells were used at passages 14 to 20. HepaRG cells were only used for testing the extracts containing or not OVTXs to investigate if any specific effect on hepatocytes can be induced, at least for the extract not containing OVTXs (OVTX-).

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Human keratinocytes HaCaT were obtained from the American Type Culture Collection (CL-00090). They were cultured in DMEM containing 5.5 mM D-glucose and 4 mM L-glutamine supplemented with 10% heat-inactivated fetal calf serum (FCS), 50 U/mL penicillin and 50 µg/mL streptomycin. Cells were used at passages 32 to 40.

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Cell treatment

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Cells were seeded in 96 well plates for cytotoxicity and IL-8 release at 10,000 cells per well for A549, EGC, HepaRG and HaCaT, at 20,000 cells per well for Caco2 and at 45,000 cells per well for Neuro2a.

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All cells were grown at 37 °C and 5% CO₂ and passaged once or twice a week depending on the cell line. The day after seeding, cells were exposed to the extracts, the fractions or the pure OVTX-a and –d for 24 hours, in the corresponding medium for each cell line but without FCS. For each cell line and experiment, a vehicle control (2% DMSO) was included. At least 3 independent experiments were performed.

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Cytotoxicity (MTT assay)

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After treatment, cells were incubated at 37 °C for 2 h with 100 µL of 500 µg/mL MTT solution prepared in medium, prior to the addition of 100 µL/well of DMSO to solubilize the mitochondrial dye. The plates were gently shaken for 5 min and the absorbance was recorded with a FLUOstar Optima microplate reader (BMG Labtek, Champigny sur Marne, France) at 570 nm. Cytotoxicity was evaluated as cell viability reduction and expressed as % cell viability compared to cells exposed to the solvent alone.

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156 Pro-inflammatory effect (IL-8 release)

157 The medium from HaCaT, Caco2, HepaRG and A549 cell cultures was collected after 24h treatment
158 and frozen at -20 °C until analysis. The release of IL-8 was measured using an enzyme-linked
159 immunosorbent assay (ELISA). TNFα (100 ng/mL) was used as positive control. 96-well microplates
160 (Nunc maxisorp) were coated with human recombinant IL-8 primary antibodies at 1 µg/mL and
161 incubated overnight at 4 °C. Between each step, the wells were washed with PBS-Tween 20 (0.05%).
162 After saturation with SuperBlock for 1 h, samples and standards (IL-8 recombinant) were added into
163 the wells and incubated at room temperature for 1.5 h. Biotin-conjugated human IL-8 antibodies (0.1
164 µg/mL) were then added followed by 100 µL of HRP 1:1000 labeling. Finally, 50 µL of the chromogenic
165 substrate TMB was added and the reaction was stopped with 50 µL sulfuric acid (1 M). Plates were
166 read with a FLUOstar Optima microplate reader (BMG Labtek, Champigny sur Marne, France) at 405
167 nm. The concentrations of IL-8 expressed in pg/mL were calculated against a standard curve prepared
168 in duplicate. Three independent experiments with two technical replicates per experiment were
169 performed.

170 Statistics

171 GraphPad Prism software (version 9.5.1) was used for IC50 calculation using the non-linear regression,  
172 sigmoïdal, 4PL model as well as for statistical analyses of IL-8 release. An analysis of the variance  
173 (ANOVA) was performed, and, when the effect of concentration was significant (p-value < 0.05), the  
174 values were compared to the control using the Dunnett's test.  
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176 **Results**

177 A) Extracts of *O. cf. ovata*

178 The two extracts differ in the number of OVTX analogs detected: 5 (OVTX-a, -b, -c, -d and -e) in MCCV54
179 extract and only 3 (OVTX-a, -d and -e) in MCCV55 extract (Gémin et al., 2022). Nevertheless, OVTX-a
180 was the major analog (around 58 and 89% of the total OVTX for MCCV54 and MCCV55, respectively).

181 The two Mediterranean strains MCCV54 and MCCV55 of *O. cf. ovata* showed a different profile of
182 OVTXs with OVTX-a being the main OVTX for both (Gémin et al. 2022). The cytotoxicity assays showed
183 a concentration-dependent effect of both extracts towards the different cell types, with a
184 concentration inhibiting cell viability by 50% (IC<sub>50</sub>) ranging from 17.3 and 91.9 ng PITX eq/mL and 2.5
185 to 31.7 ng PITX eq/mL, for MCCV54 and MCCV55 respectively (Figure 1 and Table 1).

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<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

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<caption>Figure 1: Cytotoxicity of MCCV54 (a) and MCCV55 (b) extracts on a panel of cell lines after 24 h treatment determined by the MTT assay. Mean ± standard deviation of 3 to 6 independent experiments</caption>

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192 Except the lung A549 cells that showed the highest sensitivity to the MCCV55 extract, the cells from
193 the nervous system EGC and Neuro2A cells were very sensitive to both extracts with a similar level of
194 toxicity for each cell line. However, the MCCV54 extract was 4 to 5 times less potent than the MCCV55
195 extract on these two cell lines (IC<sub>50</sub> around 20 and 4 ng PITX eq/mL respectively, Table 1).

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196 Table 1: IC<sub>50</sub> (ng PITX eq/mL) of MCCV54 and MCCV55 extracts on a panel of cell lines after 24 h
197 treatment using the MTT assay. Mean ± standard deviation of 3, 5 (*) or 6 (**) independent
198 experiments

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<table>
  <thead>
    <tr>
      <th rowspan="2">Cell type</th>
      <th colspan="2">IC<sub>50</sub> (ng PITX eq/mL)</th>
    </tr>
    <tr>
      <th>MCCV54</th>
      <th>MCCV55</th>
    </tr>
  </thead>
  <tbody>
    <tr>
      <td>A549 (lung)</td>
      <td>33.2 ± 19.4</td>
      <td>2.5 ± 1</td>
    </tr>
    <tr>
      <td>Caco2 (colon)</td>
      <td>91.9 ± 60.4 *</td>
      <td>31.7 ± 37 *</td>
    </tr>
  </tbody>
</table>
<table><tr><td>EGC (enteric nervous system)</td><td>22.7 ± 21.6 **</td><td>5.2 ± 5 **</td></tr><tr><td>Neuro2A (nervous system)</td><td>17.3 ± 10.8 *</td><td>3.3 ± 1.9 *</td></tr><tr><td>HaCaT (skin)</td><td>62.2 ± 29.8</td><td>11.8 ± 7.1</td></tr></table>

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The sensitivity of the A549 lung cells to both extracts was also very close to the ones obtained with the nervous cells (Table 1) with IC₅₀ (2.5 ng PITX eq/mL) slightly lower for the MCCV55 extract and slightly higher for the MCCV54 extract (33.2 ng PITX eq/mL). Among the cell types tested, the highest IC₅₀ was obtained with the intestinal Caco2 cell line for both extracts (IC₅₀ = 91.9 and 31.7 ng PITX eq/mL for MCCV54 and MCCV55 extracts respectively, Table 1) while the skin model gave an intermediate response (IC₅₀ = 62.2 and 11.8 ng PITX eq/mL for MCCV54 and MCCV55 extracts respectively, Table 1).

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The pro-inflammatory potential was investigated by IL-8 release after 24 h treatment with the O. cf. *ovata* extracts on A549, Caco2 and HaCaT cells. In contrast to the positive control (TNFα 100 µg/mL), no increase of IL-8 release was observed with both extracts at concentrations up to 400 µg PITX eq/mL in the A549 and Caco2 cell lines (Figure 2). Compared to the solvent control, a slight but non-significant increase of IL-8 was also observed on HaCaT cells with the MCCV55 and MCCV54 extracts (Figures 2a and 2b). However, with the MCCV54 extract, a significant increase of IL-8 was observed only at 100 ng PITX eq/mL on HaCaT cells due to the low cell viability (<5%) observed for the highest concentrations as shown in Figure 1a.

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<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

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Figure 2: IL-8 release from Caco2, A549 and HaCaT cells following a 24 h treatment with MCCV54 (a) and MCCV55 (b) extracts. Mean ± standard deviation of 3 independent experiments. Statistical significance is indicated by * p≤0.05 and **** p≤0.0001. Positive control = TNFα 100 ng/mL

## B) Fractions prepared from MCCV55 extract

Two fractions were prepared and analysed from the MCCV54 extract: one containing the different identified OVTXs as determined by LC-MS/MS called OVTX+ and one called OVTX- formed by the remaining fractions. The OVTX+ fraction (7.73 mg/mL) contain a mixture of OVTX-a (58.6%), OVTX-b (26.7%), OVTX-c (3.4%), OVTX-d (6.0%), OVTX-e (3.5%) and OVTX-f (1.8%) based on LC-MS/MS quantification (Gémin et al., 2020) and was concentrated in DMSO (Pavaux et al., 2020). The fraction OVTX+ was cytotoxic to all cell types tested (Figure 3a and Table 2). However, it could be pointed out that an increased production of formazan formation at the lowest concentrations tested was reported with the two fractions on EGC and HepaRG cells. A similar IC₅₀ was observed for the OVTX+ fraction on EGC and A549 cells (IC₅₀ = 4.4 and 7.3 ng PITX eq/mL respectively, Table 2), while the Neuro2A cells (IC₅₀ = 11.1 ng PITX eq/mL) were slightly less sensitive (2.5 fold) compared to the other nervous cell line tested EGC. Non-differentiated liver HepaRG cells were also highly sensitive to the OVTX+ fraction (IC₅₀ around 5 ng PITX eq/mL, Table 2). Moreover, as observed with the extracts, the non-differentiated Caco2 cells showed the highest IC₅₀ (15.7 ng PITX eq/mL); nevertheless, the IC₅₀ was in the same order of magnitude as for the other cell lines (Table 2).

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

Figure 3: Cytotoxicity of the fraction containing OVTX (OVTX+) (a) or not (OVTX-) (b) on a panel of cell lines after 24 h treatment using the MTT assay. Mean ± standard deviation of 3 to 4 independent experiments

With the fraction OVTX-, cytotoxicity was observed on the various cell lines tested (Figure 3b and Table 2). The EGC were the most sensitive with an IC₅₀ of 241.6 ± 71.1 ng/mL (Table 2) and a steep response between 125 and 250 ng/mL (Figure 3b). Higher and very close IC₅₀ values were obtained on both lung and liver cells (334 ± 128 and 346 ± 42 ng/mL respectively) (Table 2).

Table 2: IC₅₀ of OVTX+ and OVTX- fractions on a panel of cell lines after 24 h treatment using the MTT assay. Mean ± standard deviation of 3 or 4(*) independent experiments

<table><thead><tr><th rowspan="2">Cell type</th><th colspan="2">IC<sub>50</sub></th></tr><tr><th>OVTX+ (ng PITX eq/mL)</th><th>OVTX- (ng/mL)</th></tr></thead></table>
<table><tr><td>A549 (lung)</td><td>7.3 ± 5.3</td><td>334 ± 128</td></tr><tr><td>Caco2 (colon)</td><td>15.7 ± 2.1</td><td>387.9 ± 11.9 *</td></tr><tr><td>HepaRG (liver)</td><td>5.2 ± 0.4</td><td>346 ± 42.4</td></tr><tr><td>EGC (enteric nervous system)</td><td>4.4 ± 0.5</td><td>241.6 ± 71.1</td></tr><tr><td>Neuro2A (nervous system)</td><td>11.1 ± 1.8</td><td>498.1 ± 241</td></tr></table>

The fraction OVTX+ did not induce the release of IL-8 from lung and colon cell lines up to 100 ng PITX eq/mL (Figure 4a). However, a pro-inflammatory effect was obtained on liver HepaRG cells with a significant increase only at 6.25 ng/mL, the highest concentrations being too cytotoxic to induce pro-inflammatory effect (Figure 3a). The fraction OVTX- did not induce any pro-inflammatory effect on the intestinal Caco2 cells at any concentration tested, whereas a significant increase of IL-8 was observed at the highest concentrations (375 and 750 ng/mL) for lung A549 and hepatic HepaRG cells (Figure 4b).

<figure><img src="image_10.png" /><figcaption>Figure 4: IL-8 release from Caco2, A549 and HepaRG cells following a 24 h treatment with OVTX+ (a) and OVTX- (b) fractions. Mean ± standard deviation of 3 independent experiments. Statistical significance is indicated by * p≤0.05, ** p≤0.01 and **** p≤0.0001. Positive control = TNFα 100 ng/mL</figcaption></figure>

C) Pure OVTX-a and d and PITX
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OVTX-a and -d (Figure 5a and 5b respectively) as well as PITX (Figure 6) showed a concentration-response curve for cytotoxicity on each cell type tested. However, some differences between the responses were noticed. Globally, the IC₅₀ obtained with the 3 toxins on the different cell types ranged between 0.39 and 18.16 ng/mL (Table 3) with fold changes between 1 (for A549 cells) and 5 (for EGC).

<figure><img src="image_11.png" /></figure>

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<caption>Figure 5: Cytotoxicity of the purified OVTX-a (a) and OVTX-d (b) on a panel of cell lines after 24 h treatment using the MTT assay. Mean ± standard deviation of 3 to 5 independent experiments</caption>

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<figure><img src="image_12.png" /></figure>

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<caption>Figure 6: Cytotoxicity of PITX on a panel of cell lines after 24 h treatment using the MTT assay. Mean ± standard deviation of 3 independent experiments</caption>

The cytotoxicity assay with OVTX-a provided an IC₅₀ ranking over the panel of cell types as follows: A549<Neuro2A<EGC<HaCaT<Caco2. The IC₅₀ with OVTX-a ranged from 1.78 ng/mL for A549 cells to 18.16 ng/mL for Caco2 cells and IC₅₀ values around 2.5 ng/mL for the 3 other cell lines (Table 3). The IC₅₀ rankings for OVTX-d and PITX were slightly different: A549<Neuro2A <EGC< Caco2<HaCaT and EGC<Neuro2A< A549< Caco2<HaCaT respectively. The IC₅₀ with OVTX-d ranged from 1.24 ng/mL for A549 cells to 10.73 ng/mL for HaCaT cells and the ones for PITX ranged from 0.39 ng/mL for EGC to 7.71 ng/mL for HaCaT cells (Table 3). The A549 cells were again the most sensitive to OVTX-a and -d. The 2 nervous cell lines (i.e. EGC and Neuro2A) showed a very close IC₅₀ (around 2.5 ng/mL) for these two OVTXs (Table 3). Interestingly, the pattern was different with HaCaT keratinocytes that were more sensitive cells to OVTX-d compared to OVTX-a, while the opposite was observed for Caco-2 cells (Table 3). About PITX, the two nervous cell lines EGC and Neuro2A gave the lower IC₅₀ with very similar values (0.39 and 0.69 ng/mL respectively) while the HaCaT and Caco2 cells were less affected (IC₅₀ of 7.71 and
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6.70 ng/mL respectively (Table 3). The lung cells were rather sensitive to the 3 toxins, OVTX-a and -d showing a similar IC₅₀ to PITX, around 1.5 ng/mL.

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<table><caption>Table 3: IC₅₀ (ng/mL) of OVTX-a, OVTX-d and PITX on a panel of cell lines after 24h treatment using the MTT assay. Mean ± standard deviation of 3, 4(*) or 5(**) independent experiments</caption><thead><tr><th rowspan="2">Cell type</th><th colspan="3">IC₅₀ (ng/mL and nM)</th></tr><tr><th>OVTX-a</th><th>OVTX-d</th><th>PITX</th></tr></thead><tbody><tr><td>A549 (lung)</td><td>1.78 ± 0.97<br>0.67 ± 0.37</td><td>1.24 ± 0.03<br>0.47 ± 0.01</td><td>1.48 ± 0.44<br>0.55 ± 0.21</td></tr><tr><td>Caco2 (colon)</td><td>18.16 ± 12.20<br>6.86 ± 4.60</td><td>5.94 ± 3.53 **<br>2.23 ± 1.33</td><td>6.70 ± 2.40<br>2.50 ± 0.90</td></tr><tr><td>EGC (enteric nervous system)</td><td>2.89 ± 1.70 *<br>1.09 ± 0.64</td><td>2.51 ± 1.88 *<br>0.94 ± 0.71</td><td>0.39 ± 0.06<br>0.15 ± 0.02</td></tr><tr><td>Neuro2A (nervous system)</td><td>2.33 ± 0.31<br>0.88 ± 0.12</td><td>2.95 ± 1.72 *<br>1.11 ± 0.65</td><td>0.69 ± 0.26<br>0.26 ± 0.1</td></tr><tr><td>HaCaT (skin)</td><td>2.99 ± 1.19<br>1.13 ± 0.45</td><td>10.73 ± 3.43<br>4.03 ± 1.29</td><td>7.71 ± 3.41<br>2.88 ± 1.27</td></tr></tbody></table>

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Concentrations up to 20 ng/mL of the two ovatoxins OVTX-a and -d did not induce an increase of IL-8 release after a 24 h treatment on any of the three cell lines HaCaT, Caco2 and A549 (Figure 7a and 7b).

No pro-inflammatory effect was induced by PITX on Caco2 cells. Nevertheless, a slight, but not statistically significant, increase of IL-8 release was observed on A549 cells with PITX (Figure 7c). The only significant increase of IL-8 release following PITX treatment was detected at 5 and 10 ng/mL on HaCaT keratinocytes.
<figure><img src="image_13.png" /><figcaption>Figure 7: IL-8 release from Caco2, A549 and HaCaT cells following a 24 h treatment with OVTX-a (a) OVTX-d (b) and PITX (c). Mean ± standard deviation of 3 independent experiments. Statistical significance is indicated by * p≤0.05, ** p≤0.01, *** p≤0.001 and **** p≤0.001. Positive control = TNFα 100 ng/mL</figcaption></figure>

Discussion

In order to assess the level of toxicity of the metabolites produced by O. cf. *ovata*, we investigated the toxicity of extracts, fractions as well as purified OVTX-a and –d from two strains of O. cf. *ovata* on a panel of cell types. Due to challenges faced during the purification of OVTXs (Gémin 2020), the amount
of purified OVTX-a and -d only allowed performing *in vitro* bioassays. The responses of the different tested chemical samples on cell viability and pro-inflammatory effect using a range of cell lines were compared to the ones obtained with PITX. The biological assays on the extracts from two strains of *O. cf. ovata*, MCCV54 and MCCV55, showed that, irrespective of the cell line, the IC₅₀ was always lower for the MCCV55 extract. In fact, the MCCV54 extract contains around twice less OVTX (in PITX equivalent) than the MCCV55 extract and has also a different toxin profile including a higher percentage of OVTX-a (89 and 58% respectively) in MCCV55 compared to MCCV54 (Gémin et al. 2022). Accordingly, differences from 3 to 5 fold between the IC₅₀ of the two extracts were roughly found, except for the A549 cells where the MCCV55 extract showed an even lower IC₅₀ (2.5 ng PITX eq/mL), around 13 fold less that of the MCCV54 extract. This suggests that some metabolites more concentrated in the MCCV55 extract are strongly active on lung cells. These results are in line with a two-fold lower LD₅₀ observed for the crustacean *Artemia franciscana* exposed to the same MCCV54 and MCCV55 strains in another study (Pavaux et al. 2020).

The toxic response observed with the fraction devoid of OVTX (OVTX-) can be explained by the presence of other toxic compounds or OVTXs at concentrations lower than the LOD. A similar conclusion was raised with the same extracts and fractions of *O. cf. ovata* tested on *Artemia* (Pavaux et al. 2020). Indeed, besides OVTXs, the strain MCCV54 studied here has been recently shown to produce toxic metabolites of unidentified structure, named liguriatoxin and rivieratoxins (Ternon et al. 2022). Based on their HRMS data, none of these new compounds appear to be close PITX analogues and, on human NCI-H460 lung cells, these molecules exhibit a far lower toxicity (IC₅₀ of 680 and 3120 ng/mL respectively) compared to PITX (IC₅₀ of 0.0446 ng/mL) using the MTT assay (Ternon et al. 2022). A zwitterionic compound, ovataline, was also recently isolated from *O.cf. ovata* cultures (Lee et al. 2022) but no information on its cytotoxicity has been published so far. Ostreol A and B, isolated from a South Korean strain of *O. cf. ovata*, have been reported to induce respectively toxicity in brine shrimp (Hwang et al. 2013) and cytotoxicity on liver, neuronal and intestinal cells lines (Hwang et al. 2017). However, the *m/z* of these toxins was not detected in the MCCV54 and MCCV55 extracts by LC-MS/MS. The presence of other compounds in the two fractions OVTX- and OVTX+ could explain why there has been an increase in the metabolic activity measured by the MTT assay at the lowest concentrations tested in EGC mostly and, to some point, in HepaRG cells also (see figure 3 a and b). In fact, it has been shown with different toxic agents that formazan formation could be enhanced by increased membrane permeability, mitochondria number and activity (Ghasemi et al., 2021). Such effect can depend on the cell type as well as culture conditions (serum, medium,...), consistent with its observation only on two cell types among the 5 tested in this study.

In this study, OVTX-a and -d exhibit similar IC₅₀ values on the different cell types after a 24 h treatment, except on HaCaT cells where OVTX-d was 3.5 fold more potent than OVTX-a. The same IC₅₀ was obtained with OVTX-a and -d and with PITX on lung cells. However, PITX was slightly more potent on the two nervous cell lines (EGC and Neuro2A cells) while OVTX-a showed 2-times lower IC₅₀ on HaCaT cells after 24 h treatment. This is not in agreement with the toxicity reported previously on HaCaT human keratinocytes where a 100-fold difference between OVTX-a and PITX was described (Pelin et al. 2016b). In order to verify that this discrepancy was not due to a different protocol for cytotoxicity testing in both studies, we compared the toxicity of the 3 toxins with the same design proposed by Pelin et al (2016) that differs for the duration of the treatment (only 4 h) and a higher number of cells seeded. In this case, we did not observe any significant difference in either the response curves or IC₅₀ values for OVTX-a, OVTX-d and PITX (supplementary data, Figure S1 and Table S1). In support with our findings, the *in vivo* study of Poli et al (2018) concluded also to a close toxicity equivalence by inhalation between OVTX-a and PITX (LD₅₀ of 0.031 and 0.041 µg/kg respectively). Indeed, the isomer "AC" (for Adachi Culture) isolated by Poli et al (2018) could not be confirmed to be a derivative of OVTX-a, since no standard is currently available. Although OVTX-a and -d only differ by an oxygen in the fragment B
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(Ciminiello et al., 2010), we observed some discrepancies in their response on the different cell types that could be due to differences in affinity for the same cellular target(s) and/or differences in the mechanisms of action. Indeed, OVTX-a and -d may not act with the same affinity on the Na+/K+ ATPase, shown as the main target for PITX (Scheiner-Bobis et al. 2002; Takeuchi et al. 2008). Although OVTX-a differs by only two oxygens to PITX (Ciminiello et al. 2012), it has been shown that binding affinity of OVTX-a to HaCaT cells was twice lower than PITX (Pelin et al. 2016b). However, this result may be taken with caution as the same authors reported a 100-fold difference in toxicity on HaCaT cells that we could not confirm. Moreover, the toxins may act through other targets. For example, toxicity of PITX has been shown to affect intracellular Ca²⁺ and conductive pathway for H⁺ (Scheiner-Bobis et al. 2002; Wattenberg 2007). Nevertheless, additional studies are required to elucidate these hypotheses. The absence of difference of IC₅₀ values between PITX and OVTX-a and -d observed on A549 cells is supported by the results of acute toxicity in rats showing a very similar LD₅₀ for OVTX-a and PITX by inhalation (Poli et al. 2018).

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Only few toxicity data have been reported on OVTX-a using *in vitro* assays (Pelin et al. 2012; Poli et al. 2018; Gémin et al. 2022). As observed on HaCaT cells, inconsistent results have been obtained when comparing OVTX-a and PITX in the hemolytic test. Indeed, while Pelin et al (2016b) concluded that PITX was 10 times more active than OVTX-a, the recent study of Poli et al. (2018) did not confirm this result but described a similar IC₅₀ for the two toxins (0.43 ng/mL for PITX and 0.56 ng/mL for OVTX-a isomer AC). In contrast to OVTX-a, several *in vitro* studies have been performed with PITX on a broad range of cell types. A difference in sensitivity to PITX between the cell lines was reported, with HaCaT keratinocytes and intestinal Caco2 being the most sensitive (Pelin et al. 2018). However, we observed that Caco2 cells were less sensitive to PITX and OVTX-a and -d, among the cell types included in our study. Nevertheless, it must be emphasized that no cell lines from lung and the nervous system were tested in the previous report (Pelin et al. 2018). PITX showed an IC₅₀ for cell metabolism (Alamar blue) of 1 nM (2.6 ng/mL) after 24h exposure on rat hepatocytes clone 9 (Cagide et al. 2009), a value very close to the IC₅₀ we determined on A549 cells. In our study, the IC₅₀ for PITX on Neuro2A cells (0.87 ng/mL) was higher than the ones reported in the literature ranging from 5 pM to 170 pM (0.013 to 0.442 ng/mL) (Ledreux et al. 2009; Kerbrat et al. 2011; Nicolas et al. 2015), which is certainly due to discrepancies in the protocol (number of cells seeded, use of ouabain and veratridine).

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In this study, no pro-inflammatory effects were induced by either OVTX-a or -d, although an inflammatory effect with the same two toxins was observed on differentiated Caco2 monolayers at 5 and 1.25 ng/mL respectively (Gémin et al. 2022). The discrepancy of response observed between non-differentiated and differentiated Caco2 cells can be due to a lower number of cells and a lower IL-8 production per cell using non-differentiated cells. Indeed, the release of IL-8 obtained with the positive control (TNFa 100 ng/mL) was twice higher with differentiated Caco2 cells (Gémin et al. 2022) compared to the non-differentiated ones (this study). Nevertheless, the absence of inflammatory response following OVTX-a and -d exposure observed in this study was consistent with the absence of response on non-differentiated Caco2 cells observed with the two *O. cf. ovata* extracts as well as with the OVTX⁺ fraction. This was not the case on differentiated Caco-2 cells as MCCV54 and MCCV55 extracts induced IL-8 release (Gémin et al. 2022). Interestingly, PITX significantly increased IL-8 release in HaCaT cells as already described (Pelin et al. 2016a). Surprisingly, in A549 cells, OVTX-a and -d did not induce any pro-inflammatory effect, while PITX and the OVTX⁺ fraction induced a slight but non-significant response. This is inconsistent with the neutrophilic inflammation reported in nasal epithelium and lungs of rats exposed to PITX aerosols (Poli et al. 2018), although some discrepancy with the inflammation was reported *in vivo* in lung and liver after aerosol administration of OVTX-a (Poli et al. 2018).

399
400 **Conclusion**

401 This study provides for the first time a comparison of *in vitro* toxicity for two pure ovatoxins, OVTX-a
402 and –d, as well as *O*. cf. *ovata* extracts and fractions containing or not OVTXs over a broad panel of cell
403 types. The extracts and fractions were shown to be cytotoxic in all the cell lines tested. The fraction
404 containing OVTXs induced IL-8 release only in liver cells, while the fraction not containing OVTXs
405 induced a pro-inflammatory response both in lung and liver cells, suggesting the presence of toxic
406 compounds other than OVTXs. The pure OVTX-a and –d showed a very similar toxic potency compared
407 to PITX over several cell lines using the MTT assay. Moreover, OVTX-a and -d did not induce any pro-
408 inflammatory effect on the skin, lung and intestinal cells while PITX increased IL-8 release in
409 keratinocytes, and, to a lesser extent, on lung cells. The A549 cell model showed a rather high
410 sensitivity to OVTX-a and -d in agreement with the toxicity reported by inhalation in rats. The overall
411 results suggest that the targets of OVTX-a and -d are rather ubiquitous. If the Na+/K+ ATPase target of
412 PITX is likely shared with OVTX-a and -d, the differences in pro-inflammatory effect must be explained
413 by other mechanisms. Additional studies are required to investigate the affinity of OVTXs towards the
414 main cellular targets already described for PITX and to confirm or not their involvement in human
415 intoxications.

416

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Statements & Declarations

537

Funding

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The project OCEAN15 was funded by the French National Research Agency (Grant ANR-15-CE35-0002-04).

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Competing Interests

542

The authors have no relevant financial or non-financial interests to disclose.

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544

Author Contributions

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Conceived and designed the experiments: R.L. and V.F. Performed the experiments: R.L., ALB, L.M and M-P. G. Analyzed the data: R.L. and V.F. Wrote the paper: R.L., M-P. G., D. R. and V.F. Paper corrections: Z.A., E.T. and O.T. Funding: Z.A., O. T., E.T. and V.F.

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Data Availability

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The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Ethical Approval

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Not applicable

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Consent to Participate

All authors have participated to the project Ocean15 and consented to integrate the consortium and to work on the collaborative studies that were performed during the project.
559

560 Consent to Publish

561 All authors have approved the manuscript before submission and agree to publication.

562
**Highlights:**

* Extracts of *Ostreopsis cf. ovata* were cytotoxic to a broad range of cell lines.

* The fraction not containing ovatoxins induced IL-8 release on liver and lung cells.

* PITX was more cytotoxic than OVTX-a and -d on cells from intestine and the nervous system.

* A similar $IC_{50}$ was observed on lung cells for the three toxins.

* OVTX-a and -d did not induce IL-8 release on intestinal, lung and skin cells.

<figure><img src="image_14.png" /></figure>
**Declaration of interests**

[x] The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[ ] The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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