[go: up one dir, main page]
More Web Proxy on the site http://driver.im/
Next Article in Journal
Microcystins Exposure and the Risk of Metabolic Syndrome: A Cross-Sectional Study in Central China
Next Article in Special Issue
Lesser-Known Cyanotoxins: A Comprehensive Review of Their Health and Environmental Impacts
Previous Article in Journal
The Protein Engineering of Zearalenone Hydrolase Results in a Shift in the pH Optimum of the Relative Activity of the Enzyme
Previous Article in Special Issue
Persistence of Microcystin in Three Agricultural Ponds in Georgia, USA
You seem to have javascript disabled. Please note that many of the page functionalities won't work as expected without javascript enabled.
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Toxins
Volume 16
Issue 12
10.3390/toxins16120541
toxins-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Assessment of the Effects of Anatoxin-a In Vitro: Cytotoxicity and Uptake

by
Cristina Plata-Calzado
,
Ana I. Prieto
*,
Ana M. Cameán
and
Angeles Jos
Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González 2, 41012 Seville, Spain
*
Author to whom correspondence should be addressed.
Toxins 2024, 16(12), 541; https://doi.org/10.3390/toxins16120541
Submission received: 25 October 2024 / Revised: 2 December 2024 / Accepted: 12 December 2024 / Published: 13 December 2024
(This article belongs to the Special Issue Advances in Cyanotoxins: Latest Developments in Risk Assessment)
Browse Figures
Figure 1
<p>Reduction in tetrazolium salt MTS in (<b>a</b>) neuronal cells (SH-SY5Y and N2a), (<b>b</b>) Caco-2 and HepG2 cells and (<b>c</b>) immune system cells (THP-1, Jurkat and L5178Y Tk<sup>+/−</sup>) after 24 h of exposure to 0–200 µg/mL ATX-a fumarate. In addition, 0.3% <span class="html-italic">v</span>/<span class="html-italic">v</span> Triton X-100 was used as the positive control. All experiments were performed at least in triplicate per concentration. Data are expressed as mean ± SD compared to negative control group. Note: * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001 indicated significant difference from negative control.</p> "> Figure 2
<p>Reduction in tetrazolium salt MTS in THP-1 cells exposed to 0–3 µg/mL CYN for 24 h. In addition, 0.3% <span class="html-italic">v</span>/<span class="html-italic">v</span> Triton X-100 was used as the positive control. Experiments were performed in triplicate per concentration. Data are expressed as mean ± SD compared to negative control group. Note: * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001 indicate significant difference from the negative control.</p> "> Figure 3
<p>Chromatograms obtained by UHPLC-MS/MS of ATX-a in THP-1 cells exposed to 50 µg/mL ATX-a fumarate for 24 h. (<b>a</b>) Negative control, (<b>b</b>) intracellular fraction and (<b>c</b>) extracellular fraction.</p> "> Figure 4
<p>Effects of ATX-a on the expression of mRNA of genes involved in apoptosis/necrosis in THP-1 cells. Cells were exposed to 5 or 50 µg/mL ATX-a fumarate for 24 h. CPT (0.5 µM) was used as positive control and chloroform/methanol (4:1) as solvent control. Results are expressed as relative mRNA expression normalized to the negative control group. * <span class="html-italic">p</span> &lt; 0.05 and **** <span class="html-italic">p</span> &lt; 0.0001 indicate significantly difference from negative controls.</p> ">
Versions Notes

Abstract

:
Anatoxin-a (ATX-a) is a cyanotoxin whose toxicological profile has been underinvestigated in comparison to other cyanotoxins such as microcystins (MCs) or cylindrospermopsin (CYN). However, its wide distribution, occurrence, and toxic episodes justify more attention. It is classified as a neurotoxin, but it has also been reported to affect other organs and systems. Thus, the aim of this study was to establish, as a first tier in its toxicological evaluation, its cytotoxicity in a wide range of cell lines representative of potential target organs (N2a, SH-SY5Y, HepG2, Caco2, L5178Y Tk+/−, THP-1 and Jurkat). As limited effects were observed after exposure to up to 200 µg/mL of ATX-a for 24 h (only Jurkat and THP-1 cells showed reduced cell viability), cell uptake experiments were performed by ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). The results showed that the immune system cells had the highest percentage of ATX-a in the intracellular fraction, followed by neuronal cells and finally Caco-2 and HepG2 cells. Moreover, the expression of genes related to cell death mechanisms in THP-1 cells was also analyzed by polymerase chain reaction (PCR) and showed no changes under the conditions tested. Further research is required on ATX-a’s toxic effects and toxicokinetics to contribute to its risk assessment.
Keywords:
anatoxin-a; cytotoxicity; in vitro; uptake; gene expression; risk assessment
Key Contribution: ATX-a only induced cytotoxicity in THP-1 and Jurkat cells at high concentrations. ATX-a was detected inside the established cell lines at different levels. No relationship between cytotoxicity and toxin uptake was observed. ATX-a did not change apoptosis/necrosis-related gene expression in THP-1 cells.

1. Introduction

Cyanobacterial blooms can pose a risk to the environment and public health as a result of their ability to produce cyanotoxins. Factors such as climate change and pollution from anthropogenic activity can lead to an increase in the frequency of such blooms. Therefore, the investigation of the toxic effects produced by cyanotoxins such as microcystins (MCs), cylindrospermopsin (CYN) or anatoxin-a (ATX-a) is a priority [1,2]. Of all these cyanotoxins, ATX-a is the least studied. According to the World Health Organization (WHO), more toxicological data are needed in order to update the provisional reference values established for ATX-a [3]. ATX-a is a secondary amine alkaloid produced by numerous cyanobacteria genders such as Anabaena, Aphanizomenon, Oscillatoria, or Microcystis [4].
ATX-a has been reported in Africa [5,6], Asia [7,8], Europe [9,10,11,12], America [13,14] and Oceania [15,16]. Furthermore, its presence is not limited to freshwater, but several studies have reported ATX-a in brackish water [17,18]. In terms of detected concentrations of ATX-a, this cyanotoxin typically remains below 10 µg/L in open water [3], although concentrations higher than 1000 µg/L have been documented in surface water [19]. Moreover, Wood et al. [20] detected up to 100-fold variations in the amount of ATX-a in toxic strains isolated from the same mat. These findings reveal that there is a large variability in the ATX-a content in different environmental samples, so toxic effects also need to be investigated in a wide concentration range.
Regarding its toxicity, it is important to note that several studies have described animal poisonings, mainly in dogs, due to exposure to this cyanotoxin [21,22,23,24,25]. In humans, the routes of exposure to ATX-a include inhalation, dermal, and oral routes, with the latter being the most relevant due to the consumption of contaminated water or food (mainly fish and vegetables) [26]. In this regard, human intoxication has recently been described as the result of the ingestion of ATX-a-contaminated sea figs [27], as well as the presence of ATX-a in bivalve samples at the Ingril site and at the Le Scoré site [28]. The results of these studies highlight the need to carry out ATX-a toxicity studies to clarify the consequences of the exposure to this cyanotoxin.
Early toxicity studies of ATX-a in isolated organs focused on demonstrating the mechanism of action, observing a high isomer-dependent affinity for nicotinic acetylcholine receptors (AChR) [29,30,31], with the (+)-ATX-a isomer being more than 100 times more potent than the (−)-ATX-a isomer [30]. Binding of ATX-a to AChR produces a neuromuscular blockade that results in symptoms such as fatigue, convulsions, paralysis, and respiratory failure, among others [3]. For this reason, ATX-a is classified mainly as a neurotoxin. Nevertheless, few studies have explored the toxic effects of this cyanotoxin using neuronal cell lines and have shown conflicting results. Thus, Takser et al. [32] studied the toxic effects of ATX-a on the N2a cell line. This study showed a significant reduction in N2a viability compared to the control group by MTT assay after exposure for 24, 48 and 72 h to 0.1 and 10 µM ATX-a. However, Kubickova et al. [33] did not observe ATX-a cytotoxicity in any of the assays performed (neutral red uptake, reduction in resazurin, lactate dehydrogenase release, acetoxymethyl ester cleavage and carboxyfluorescein diacetate) in human neural stem cells derived from the H9 cell line after exposure to concentrations of up to 20 µM ATX-a for 4 days.
In addition, interestingly, the effects of ATX-a on other tissues and systems have been reported. Thus, ATX-a has been shown to exhibit immunotoxic [32,34,35,36,37], cytotoxic [38,39], genotoxic [40] or cardiovascular effects [41,42,43]. However, most of these studies have been carried out on isolated organs or cells or used in vivo models.
Overall, although in vitro studies are one of the first steps in the toxicity evaluation of compounds, studies focused on the toxic effects of ATX-a in established cell lines are limited. Therefore, the aim of the present study was to investigate the potential cytotoxicity of ATX-a in different cell lines as the first tier in the process to elucidate its toxicological profile. Specifically, neuronal cell lines (N2a, SH-SY5Y), immune system cell lines (THP-1, Jurkat and L5178Y Tk+/−), and cell lines representative of an oral exposure (HepG2 and Caco-2) were employed, in which the possible cytotoxic effects of this cyanotoxin have been scarcely or not yet explored. Furthermore, in order to correctly interpret the results obtained in cell viability assays, the ATX-a uptake by the seven cell lines used was also analyzed, and for comparative purposes, CYN cytotoxicity and uptake in THP-1 cells were also included. Finally, the effects on the expression of genes related to apoptosis and necrosis mechanisms were explored in the cell line in which the highest cytotoxic effect of ATX-a was observed (THP-1 cell line).

2. Results

2.1. Cytotoxicity Assays

The results obtained in the cytotoxicity assays with ATX-a are shown in Figure 1. A non-significant reduction in viability was observed in neuronal cells (SH-SY5Y and N2a) after 24 h of exposure to 0–200 µg/mL of ATX-a fumarate (equivalent to 0–117.5 µg/mL pure ATX-a) (Figure 1a). Similar results were obtained in the Caco-2 and HepG2 cell lines after the same exposure conditions (Figure 1b). A decrease in viability of the Jurkat cell line was observed after exposure to ATX-a fumarate for 24 h at all tested concentrations (Figure 1c). However, a significant decrease in cell viability was only obtained in the THP-1 cell line after exposure to the maximum concentration tested (200 µg/mL) compared to the control group.
As for CYN, a reduction in cell viability was observed at all concentrations employed. Specifically, an EC20 was obtained in THP-1 cells after exposure to 1.1 µg/mL of toxin for 24 h. The other concentrations assayed (2.5 and 3 µg/mL CYN) showed a viability of THP-1 cells 69.05 ± 8.7% and 57.67 ± 3.5%, respectively, compared to the negative control (Figure 2).

2.2. Uptake of ATX-a

An analysis of ATX-a uptake by the cell lines used in the cytotoxicity assays was performed by UHPLC-MS/MS. The limits of detection (LOD) and quantification (LOQ) of the analytical method were 1.88 and 6.28 µg/L, respectively. As an example, ATX-a uptake chromatograms in the THP-1 cell line after exposure to 50 µg/mL ATX-a fumarate for 24 h are shown (Figure 3). Chromatograms corresponding to the other six cell lines exposed to the same concentration of ATX-a fumarate are available in the Supplementary Material (Figures S1–S3).
The results obtained in the determination of ATX-a in the cell lines assayed are shown in Table 1. In this way, taking into account the amount of toxin added, between 43.55 and 129.84% of ATX-a was detected in the analysis. The results showed ATX-a in the intracellular fraction of all cells analyzed. However, the amounts detected in each of the cell types were different. Specifically, a higher uptake was observed in the following cell lines belonging to the immune system: L5178Y Tk+/− (6.45–9.70%), Jurkat (10.58–13.96%) and THP-1 (6.62–14.72%) of total ATX-a added, followed by neuronal cell lines (between 1.52 and 2.94%) and finally, in Caco-2 and HepG2 (between 0.30 and 0.65% of total toxin added) (Table 1).

2.3. Uptake CYN

The results of CYN uptake in the THP-1 cell line expressed as % of CYN detected with respect to the total amount of toxin added are shown in Table 2. The percentages of detected CYN ranged from 57.81 to 65.85%. An increase in the percentage of intracellular CYN was observed as the concentration of exposure to this cyanotoxin increased.

2.4. Gene Expression Analysis by Quantitative Real-Time PCR

The effects of ATX-a exposure for 24 h on mRNA expression of genes involved in cell death mechanisms were analyzed in the THP-1 cell line by RT-qPCR (Figure 4). ATX-a did not produce changes in the expression of the genes investigated at either of the two concentrations assayed (5 and 50 µg/mL ATX-a fumarate) under the conditions tested. Changes in gene expression were only observed after exposure to the positive control: upregulation in BAX and downregulation in BCL2.

3. Discussion

Although ATX-a is a well-known cyanotoxin, the amount of studies and experimental data dealing with it is lower compared to other cyanotoxins such as microcystins [44] or cylindrospermopsin [45], as it was evidenced by the review performed by Plata-Calzado et al. [46]. However, it is a widely reported toxin in important cases of deaths of dogs, livestock and wild animals poisoning, with convulsions, ataxia and paresthesia of cerebral hypoxia being some of the main toxic effects observed after ATX-a exposure [3]. In addition, the WHO pointed out that the toxicological database on ATXs was not adequate to support derivation of a formal guideline value [3]. Thus, to further explore the mechanisms of ATX-a toxicity, cell viability studies with different established cell lines were performed. These assays are a basic approach in toxicity-testing strategies and could support the establishment of the adverse outcome pathways (AOPs) of the studied chemical. As ATX-a is primarily classified as a neurotoxin, two different neuronal cells, SHSY-5Y and N2a, were first selected. Both have been reported to express AChR, of which ATX-a is an agonist [47,48]. Surprisingly, no evident cytotoxicity was observed at low concentrations on SH-SY5Y, but even at high concentrations (up to 200 µg/mL) in both cell lines. On the contrary, CYN, although not specifically classified as a neurotoxin, was reported to induce a higher cytotoxicity on SHSY-5Y cultures, with an EC50-24h value of only 0.87 ± 0.13 µg/mL using the same assay [49]. The number of studies of ATX-a in neuronal stablished cell lines is limited. Only Takser et al. [32] reported cytotoxicity in N2a cells after exposure to 0.1 and 10 µM ATX-a (equivalent to ≈0.028 and 2.8 µg/mL, respectively) in the MTT assay, although the effects were of similar magnitude, without a concentration-dependent pattern. In addition, an EC50 was only reached after 72 h of exposure to ATX-a. In contrast, these ATX-a concentrations did not induce cytotoxicity in BV-2 cells (murine microglial cells).
As cyanotoxins are reported to induce toxicity at different levels, other established cell lines of additional target organs were selected to test ATX-a in order to check cytotoxicity responses. Thus, HepG2 and Caco-2 cells were used taking into account that oral exposure is the main exposure for cyanotoxins in the general population. But results provided a similar outcome, no evident cytotoxicity. To our knowledge, no studies on the possible in vitro adverse effects of ATX-a on the gastrointestinal system have been published, although alterations in enzyme biomarkers have been detected in fish liver after intraperitoneal administration of ATX-a [50].
Additionally, cells lines related to the immune system (monocytes and lymphoblasts) were selected. In this case, L5178Y Tk+/− cells did not experience toxic effects, Jurkat cells reduced its viability in a non-significant way, and THP-1 cells significantly reduced its viability at the highest concentration tested (200 µg/mL). Scientific literature reports toxic effects of ATX-a on the immune system. Thus, Takser et al. [32] observed cytotoxicity in the RAW246.7 murine macrophage-like cell line exposed to 0.1 and 10 µM ATX-a. Moreover, ATX-a has shown its toxicity on immunological models from fish [36,37] but using isolated cells and not established cell lines, making the comparison more difficult. Regarding mammalian models, apart from Takser et al. [32], the most relevant studies to compare with would be those by Rao et al. [34] and Teneva et al. [35]. Rao et al. [34] exposed thymocytes isolated from 4- to 8-week-old rats to (+)-ATX-a and observed a strong concentration-dependent decrease in cell viability with a total reduction at 50 µg/mL. Teneva et al. [35] also observed a significant cytotoxicity in spleen cells isolated from male 8-week-old BALB/c mice exposed to 0.1 µg/mL for 24 h, with no specification of the isomer used. These differences observed with respect to our study could be due to the ATX-a isomer used. Thus, Zhang et al. [30] found that (+)-ATX-a was 160-fold more potent than (−)-ATX-a, and 20-fold more potent than (±)-ATX-a in inhibiting [3H]ACh binding in rat brains. However, Molloy et al. [31] used synthetic (±)-ATX-a (up to 100 µg/mL) in bovine adrenal chromaffin cells showing that ATX-a was a potent nicotinic acetylcholine receptor agonist in these cells. Similarly, Liu et al. [51] also used the racemic mixture, and they reported agonist activity in the yeast oestrogen screen assay in Saccharomyces cerevisiae. In our study, the concentrations of (+)-ATX-a contained in the racemic mixture exceed those used by Rao et al. [34] and Teneva et al. [35], so the experimental model (primary culture versus established cell line) could be the main reason for the differences found with respect to our results.
Thus, considering the cytotoxicity data obtained from a general point of view, it has been evidenced that ATX-a showed a limited cytotoxicity in the established cell lines tested, and that the type of experimental model used could play a role. However, Takser et al. [32] used the same cell line (N2a), a similar assay (MTT assay, based also on the mitochondrial enzyme succinate dehydrogenase activity), and the same toxin (ATX-a from Enzo) and they observed cytotoxicity. More recently, Kubickova et al. [33] have tested ATX-a using a differentiating in vitro human neural stem cell model and observed that ATX-a (up to 20 µM) did not cause distinct developmental neurotoxicity alone, and that its (excito-)toxicity to acetylcholinergic neurons occurred only in co-exposure to all-trans retinoic acid.
The results could also be explained due to the non-effective uptake of ATX-a by the cells. Thus, other widely studied cyanotoxins, such as MCs [2], are known to require specific transporters to enter the cells. They are uptaken by the organic acid transporter polypeptides (OATPs) and limited toxicity on established cell lines has been explained by the lack of these specific transporters [52]. Unfortunately, data regarding ATX-a kinetics are limited, and no reports suggest the participation of transporters. Thus, Testai [2] stated that ATX-a is passively and rapidly absorbed after ingestion and widely disseminated to different tissues, including the brain. Thus, to check the uptake of ATX-a by the cells, the determination of the toxin was performed in the supernatants and inside the cells after 24 h of exposure to the toxin. The results showed that the uptake in all cell lines was limited. Cells of immunological nature showed a higher intracellular presence than those from the nervous system and from the gastrointestinal system. This was in agreement with the cytotoxicity results. Also, an interesting observation derived from the data obtained is that the uptake (intracellular recovery) was concentration-dependent in all cell lines, with the exception of the THP-1 and HepG2 cell lines, where the amount of toxin detected in the intracellular fraction was similar at both concentrations. Therefore, at least a small part of ATX-a could be taken up by passive diffusion, which would explain why it is detected inside all cell lines and that the amount was concentration-dependent. In addition, the main aim of the present work was to analyze whether the content of uptaken toxin was relevant to induce cytotoxicity. However, the results do not seem to show a relationship between uptake and the observed toxic effects, and with that aim, a similar experiment with CYN was performed for comparative purposes. Thus, cytotoxicity-causing CYN concentrations were used, and the percentage of cytotoxicity and uptake was analyzed. The cytotoxicity assays performed by Casas-Rodriguez et al. [53] were replicated in the THP-1 cell line, obtaining similar results (EC20 = 1.1 µg/mL and approximately EC50 at a concentration of 3 µg/mL CYN). Regarding toxin uptake, a concentration-dependent increase in intracellular % was also observed. However, the % intracellular recovery was very low and even much lower than in the case of ATX-a, and despite this, an evident cytotoxicity was observed. Therefore, the absence of ATX-a cytotoxicity is not only a consequence of low toxin uptake.
A different hypothesis to try to explain the absence of effects is that cytotoxicity is a non-very sensitive biomarker of cell damage, and very high concentrations are required. This argument was used by Menezes et al. [54] for the toxic effects induced by MC-LR in the kidney Vero-E6 cell line. They concluded that the type and extent of effects were largely influenced by the toxin concentration, noting that certain responses, like cell death by apoptosis or necrosis, need much higher concentration compared to others, such as the expression of mitogen-activated protein kinase (MAPK). In order to test this hypothesis, the expression of genes involved in cell death was analyzed in the cell line most sensitive to cytotoxic effects, THP-1 cells. Also, it is known that for other cyanotoxins, such as CYN, low concentrations (EC20 values) can already induce important changes at a genetic level [53]. However, in this case, again, no effects were observed. Nevertheless, we cannot dismiss the possibility that the lack of direct cytotoxicity of ATX-a could imply a potential ATX-a toxicity mediated by other responses such as interactions with certain molecular receptors or altering cellular processes that do not result in immediate cell death. On the other hand, in comparison to CYN, it might happen that the limited in vitro cytotoxicity observed for ATX-a is just a matter of toxic potency. Thus, CYN has proved to be more cytotoxic than ATX-a on the different cell lines studied [49,53,55,56]. Regarding in vivo data, median lethal dose values after acute exposure reported by the WHO [3,57] were lower for CYN than for ATX-a, but a direct comparison is not possible due to differences in exposure, purity and concentrations of toxin used, etc.
Overall, the results obtained in this research reveal important issues regarding the selection of the most suitable experimental models and biomarkers to use in ATX-a toxicological research. However, the observation that ATX-a is taken up by all cell lines tested suggests that this toxin can potentially affect various tissue types and that there is no cytotoxic potential in these cell models under the conditions tested. In view of the results obtained, further research with more advanced and sensitive experimental models is needed to elucidate their possible absorption pathways and toxicity mechanisms, in order to clarify their toxic potential to support an adequate risk assessment.

4. Conclusions

The cytotoxicity of ATX-a in the cell lines evaluated was low even at high concentrations (200 µg/mL). This may be due to different reasons among which the experimental model, isomer specificity of toxin, toxin uptake or the cell damage marker used stand out. The ATX-a uptake results showed toxin inside all the cell lines studied, concluding that the absence of cytotoxicity was not due to a limited uptake. In contrast, CYN with a lower uptake induced higher cytotoxicity. Also, no response was observed in the expression of genes related to the apoptosis and necrosis response. The results seem to indicate that the established cell lines might not be a suitable model to study ATX-a toxicity, and that ATX-a toxic potency is lower than for CYN. Thus, although the established cell lines are a first step in the investigation of the toxic effects of substances, the present study shows that in the case of ATX-a, common cell models such as N2a, SH-SY5Y, THP-1, Jurkat, L5178Y Tk+/−, HepG2 and Caco-2 cell lines were not the most appropriate. Studies with more advanced models such as primary cell cultures or in vivo systems are needed to better understand the mechanisms and toxic effects of ATX-a.

5. Materials and Methods

5.1. Chemicals

(±) Anatoxin-a fumarate standard with a purity greater than 98.0% and cylindrospermopsin with a purity of 95% were provided by Enzo Life Sciences (Lausen, Switzerland). The reagents needed for cell culture were sourced from Gibco (Biomol, Sevilla, Spain). Reagents for cyanotoxin extraction and LC-MS/MS analysis were obtained from Sigma–Aldrich (Madrid, Spain). Finally, the chemical reagents used for RT-qPCR were provided by Qiagen (Madrid, Spain) and Bio-Rad Laboratories (Hercules, CA, USA).

5.2. Cell Lines and Culture Conditions

The cell lines as well as the culture conditions used are detailed in Table 3. The recommendations of the supplier of the cell lines (the American Type Culture Collection (ATCC)) for their maintenance were followed.

5.3. Test Solutions

Stock solutions of ATX-a fumarate (1000 µg/mL) and CYN (1000 µg/mL) were prepared in milli-Q sterile water and maintained at −20 °C. Toxin concentrations were prepared in the corresponding cell culture medium.

5.4. Cytotoxicity Assays

For cytotoxicity assays, cells were seeded at different densities depending on the growth of each cell line in 96-well culture plates and following the supplier’s recommendations. Thus, N2a, SH-SY5Y and L5178Y Tk+/− were seeded at 2 × 105 cells/mL, THP-1 and HepG2 at 3 × 105 cells/mL, Jurkat at 5 × 105 cells/mL and Caco-2 at 7.5 × 105 cells/mL. Cells were incubated at 37 °C and 5% CO2 for 24 h until 70% confluence was reached. Subsequently, cells were exposed to concentrations of ATX-a fumarate ranging from 0 to 200 µg/mL (equivalent to 0–117.5 µg/mL pure ATX-a). Previously, preliminary cytotoxicity assays were performed on the SH-SY5Y cell line with lower concentrations of ATX-a fumarate (2.5, 5, 7.5, 10, 15, 20, 40, and 60 µg/mL) for 24 h. No decrease in cell viability was observed compared to the control group at those concentrations, so higher concentrations were considered for further cytotoxicity assays (up to 200 µg/mL ATX-a fumarate). The limit of 200 µg/mL was established taking into account the data reported on the highest ATX-a concentrations found in nature (1000 µg/L) [19]. To check appropriate test conditions, Triton X-100 (0.3% v/v) with a recognized cytotoxic effect was introduced into the assay as a positive control. The cells were treated with the corresponding concentration for 24 h. The biomarker assayed was the reduction in the tetrazolium salt MTS (3-(4,5-dimethylthiazol-2-yl)5-(3-carbox-ymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium salt) assay following the instructions of the manufacturer’s protocol for the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madrid, Spain). After 2 h of incubation with MTS, the absorbance was measured at 492 nm by absorption spectrophotometry. Cell viability obtained at the exposure concentrations was expressed as % with respect to the negative control.
As ATX-a did not reduce cell viability to a high extent, a parallel cytotoxicity assay with CYN was performed in THP-1 cells following the same protocol to compare the cytotoxicity produced with the uptake of both toxins by this cell line, since they both are hydrophilic cyanotoxins that can be found in nature simultaneously [22]. Therefore, cells were exposed to concentrations of CYN ranging from 0 to 3 µg/mL to obtain effective concentration values (EC20 and EC50), according to previously published results [53].

5.5. Evaluation of ATX-a Uptake

5.5.1. Exposure

The cell treatment was the same as described above for cytotoxicity assays (Section 2.4) but with ATX-a fumarate concentrations of 50 and 100 µg/mL (equivalent to 29.4 and 58.7 µg/mL ATX-a). After exposure time, cells and medium were collected separately and stored at −20 °C until further analysis. For adherent cells, after collecting the medium, 0.05% (1X) trypsin-EDTA was added to lift the cells and stored. For suspension cells, cells were centrifuged 5 min at 2000× g rpm, the supernatant was collected, and the cells were resuspended in 50 µL of fresh medium and stored at −20 °C. All exposure treatments were performed by triplicate.

5.5.2. Extraction and Purification Procedures

The ATX-a content was extracted following the protocol of Dimitrakopoulus et al. [58]. To extract the toxin, 5 mL of 20% methanol (MeOH) was added to the cell samples and the supernatants and sonicated for 10 min. Then, samples were centrifuged at 3700× g rpm for 30 min. The supernatant was collected, and the pH was adjusted to 10.5 with NaOH. Briefly, graphitized carbon cartridges (Bond Elut cartridges) supplied by Agilent Technologies (Santa Clara, CA, USA) were used for solid-phase extraction (SPE). The SPE cartridge was preconditioned with 6 mL of MeOH and equilibrated with 6 mL of water. Subsequently, the sample at pH 10.5 was passed through the cartridges and eluted with 6 mL of MeOH + 0.1% trifluoroacetic acid. The resulting extracts were evaporated to dryness in a rotary evaporator and resuspended in 1 mL MeOH 20% prior to analysis by ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS).

5.5.3. UHPLC-MS/MS

In order to carry out the determination of ATX-a, the possible interferences that can be caused by the amino acid phenylalanine have been taken into account [59]. Thus, previous analyses were performed with a standard of ATX-a and phenylalanine, with the aim of verifying that the chromatographic conditions allowed the correct differentiation of both compounds. Therefore, ATX-a determination was carried out using a Thermo Scientific liquid chromatography system consisting of a binary UHPLC Dionex Ultimate 3000 RS, connected to a quadrupole-orbitrap QExactive hybrid mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) with a HESI ionization probe. UHPLC analyses were performed on a 100 × 2.1 mm Acquity HSS T3 1.8 µm column. The flow rate was 0.450 mL/min. A gradient of (A) water and (B) acetonitrile, each containing formic acid (FA) (v/v) in a proportion of 0.1%, was achieved. An injection volume of 5 µL was also set. The profile of elution was as follows: 2% B for 0.8 min, then 70% B for 9 min, 80% B for 1 min, and the last 2 min with 2% B. A Parallel Reaction Monitoring (PRM) method was employed in positive mode. The HESI source parameters were sheath and auxiliary gas flow rates, 60 and 25 arbitrary units, respectively; spray voltage, 3.5 kV; capillary temperature, 320 °C; S-lens RF level, 50; and probe heater temperature, 430 °C. The PRM method was used at a resolution of 70,000 at m/z 200 full width at half maximum (FWHM), and the monitored pseudomolecular ion [M+H]+ had a m/z of 166.12264.
In addition, ATX-a uptake calculations took into account the amount of toxin present in the reference standard used (ATX-a fumarate), according to the following relation: Corrected mass = (MWanatoxin-a/MWanatoxin-a fumarate) × Measured mass.

5.6. Evaluation of CYN Uptake

5.6.1. Exposure, Extraction and Purification Procedures

Treatment of THP-1 cells with CYN concentrations of 1.1, 2.5 and 3 µg/mL was performed as described above for cytotoxicity assays (Section 5.4). After 24 h of exposure, cells were centrifuged 5 min at 2000× g rpm, the supernatant was collected, and the cells were resuspended in 50 µL of fresh medium and stored at −20 °C, following the same methodology as for ATX-a uptake assessment.
After that, CYN was extracted using the protocol validated by Guzmán-Guillén et al. [60]. Firstly, to extract the toxin, 3 mL of milliQ water was added to the cell samples and the supernatants and sonicated for 15 min. Then, samples were stirred for 1 h and sonicated 15 min more. Subsequently, the samples were centrifuged at 3700× g rpm for 10 min. In addition, 6 µL trifluoroacetic acid was added to the supernatant. Finally, it was shaken for 1 h and stood for 3 h.
Regarding clean-up, graphitized carbon cartridges (Bond Elut cartridges) were used for SPE. The SPE cartridge was preconditioned with 10 mL of DCM/MeOH (10/90) with 5% FA and then equilibrated with 10 mL of milliQ water. Subsequently, the sample was passed through the cartridges, washed with 10 mL of milliQ water and eluted with 10 mL of the acidified DCM/MeOH (10/90) solution. The resulting extracts were evaporated to dryness using a rotary evaporator and resuspended in 500 µL milliQ water before analysis by UPLC-MS/MS analysis.

5.6.2. UPLC-MS/MS Determination of CYN

An UPLC Acquity instrument (Waters, Milford, MA, USA) was used to perform the chromatographic separation. This equipment was connected to a Xevo TQS-micro instrument (Waters, Milford, MA, USA), which consists of a triple quadrupole mass spectrometer, and utilized an electrospray ionization source operation in positive mode. The separation was conducted on an Acquity BEH C18 column (50 × 2.1 mm, 1.7 µm particle size) with a flow rate of 0.45 mL/min. The mobile phase consisted of (A) water containing 0.1% FA (v/v) and (B) methanol with 0.1% FA (v/v). The gradient elution profile was programmed as follows: 0% B for 0.8 min, followed by a linear increase to 90% B over 2.2 min, maintained at 90% B for 1 min, and finally returned to 0% B for 1 min. The injection volume was set at 5 µL. For detection, Multiple Reaction Monitoring (MRM) was employed, with parent ions monitored in Q1 and fragments ions in Q3. For quantification, the transition of CYN used was 416.2/194.0 and for confirmation, it was 416.2/176.0. The mass spectrometer parameters were optimized as follows: capillary voltage at 3.0 kV, desolvation gas flow at 1000 L/h, source desolvation temperature at 500 °C, and cone gas flow at 50 L/h.

5.7. Real-Time Quantitative PCR (qRT-PCR) Analysis After ATX-a Exposure

For gene expression analysis, THP-1 cells were selected because they showed a higher cytotoxicity of the toxin. Cells were seeded at 5 × 105 cells/mL and incubated for 24 h at 37 °C in 5% CO2. Subsequently, cells were exposed to fresh medium containing 5 and 50 µg/mL ATX-a fumarate and incubated for 24 h. The concentrations were selected according to the concentrations found in nature (equivalent to 5 µg/mL ATX-a fumarate) [61,62] and for comparison against the results obtained in the rest of tests (50 µg/mL ATX-a fumarate). Although these concentrations did not decrease cell viability, alteration of gene expression is a molecular event induced at lower concentrations than cytotoxicity [52,54].
Cell medium was used as a negative control and camptothecin (CPT) (Sigma-Aldrich, C9911, Madrid, Spain) at 0.5 µM dissolved in chloroform/methanol (4:1) was used as a positive control. For this reason, a solvent positive control (chloroform/methanol 4:1) was introduced in the assay. After ATX-a treatment, cells were centrifuged at 300× g for 5 min and resuspended with 1 mL of fresh medium. RNA was extracted according to the manufacturer’s instructions for the RNAeasy Mini Kit (Cat: 74104, Qiagen, Madrid, Spain). In addition, RNA purification was carried out using the RNAse-free DNAse set (Cat: 79254). After that, RNA purity was measured using the ratio 260/280 nm and 260/230 nm in NanoDrop 2000 (Thermo Scientific, Pittsbutgh, PA, USA). The samples were stored at −80 °C until reverse transcription was performed. In addition, 1 µg of total RNA was used to obtain cDNA by reverse transcription (RT) using the QuantiTec® reverse transcription kit (Cat: 205311, Qiagen, Madrid, Spain) according to the supplier’s instructions. Samples were kept at −20 °C until quantitative real-time polymerase chain reaction assay (RT-qPCR) was performed. The expression of apoptosis/necrosis genes including B-cell lymphoma 2 (BCL2), Bcl-2-associated X protein (BAX) and receptor-interacting Serine/Threonine-Protein Kinase 3 (RIPK3) were analyzed. Also, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene was used for the calculations. The primers used are shown in Table S1. A 1:5 dilution of cDNA in RNAse-free water was performed and amplified by PCR in a 384-well plate. Diluted cDNA, the prime PCR probe (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for the corresponding gene and the iTaq universal probes Supermix (Cat: 1725134) were used for the reaction in a final volume of 10 µL. Amplification was performed using the Light Cycler®480 System (Roche, Berlin, Germany) with an initial denaturation step at 95 °C for 2 min followed by 50 cycles of 95 °C for 5 seg and 60 °C for 30 seg. The results were determined using the 2−ΔΔCT method.

5.8. Statistical Analysis

The distribution of the data was checked with the Kolmogorov–Smirnov test. For statistical analysis, data with a normal distribution were analyzed using the analysis of variance (ANOVA) followed by Dunnett’s multiple comparison. For data that did not follow a normal distribution, the Kruskal–Wallis test was employed, followed by Dunn’s multiple comparison tests. All results were analyzed with Graph-Pad Prism 8.0.1 Software (Graph-Pad Prism 8 Software Inc., La Jolla, CA, USA). The results were expressed as mean ± standard deviation (SD). Differences were considered significant at * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, respectively.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/toxins16120541/s1: Figure S1: Chromatograms obtained by UHPLC-MS/MS of ATX-a in L5178YTk+/− and Jurkat cells exposed to 50 µg/mL ATX-a fumarate for 24 h. (a) Negative control, (b) intracellular fraction; (c) extracellular fraction. Figure S2: Chromatograms obtained by UHPLC-MS/MS of ATX-a in Caco-2 and HepG2 cells exposed to 50 µg/mL ATX-a fumarate for 24 h. (a) Negative control, (b) intracellular fraction; (c) extracellular fraction. Figure S3: Chromatograms obtained by UHPLC-MS/MS of ATX-a in N2a and SH-SY5Y cells exposed to 50 µg/mL ATX-a fumarate for 24 h. (a) Negative control, (b) intracellular fraction; (c) extracellular fraction. Table S1: Primers used for PCR reactions in this study for responses to apoptosis (BAX, BCL-2), and necrosis (RIPK3) and the housekeeping gene (GAPDH).

Author Contributions

Conceptualization, C.P.-C., A.I.P. and A.J.; methodology, C.P.-C., A.I.P. and A.J.; formal analysis, C.P.-C. and A.I.P.; investigation, C.P.-C., A.I.P., A.M.C. and A.J.; resources, A.M.C. and A.J.; data curation, C.P.-C., A.I.P. and A.J.; writing—original draft preparation, C.P.-C. and A.J.; writing—review and editing, C.P.-C., A.I.P., A.M.C. and A.J.; visualization, C.P.-C., A.I.P., A.M.C. and A.J.; supervision, A.I.P., A.M.C. and A.J.; project administration, A.I.P., A.M.C. and A.J.; funding acquisition, A.I.P., A.M.C. and A.J. All authors have read and agreed to the published version of the manuscript.

Funding

Project PID2019-104890RB-I00 funded by MICIU/AEI/10.13039/501100011033 and Project PID2023-147444OB-I00 funded by MICIU/AEI/10.13039/501100011033 and FEDER, UE. C.P.-C. thanks the Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía for awarding the predoctoral contract (PREDOC_00447) in the Predoctores 2021 grant.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to acknowledge the Project PID2019-104890RB-I00 funded by MICIU/AEI/10.13039/501100011033 and Project PID2023-147444OB-I00 funded by MICIU/AEI /10.13039/501100011033 and FEDER, UE. C.P.-C. thanks the Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía for awarding the predoctoral contract (PREDOC_00447) as part of the Predoctores 2021 grant.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Buratti, F.M.; Manganelli, M.; Vichi, S.; Stefanelli, M.; Scardala, S.; Testai, E.; Funari, E. Cyanotoxins: Producing organisms, occurrence, toxicity, mechanism of action and human health toxicological risk evaluation. Arch. Toxicol. 2017, 91, 1049–1130. [Google Scholar] [CrossRef]
  2. Testai, E. Cyanobacterial toxins. In Toxic Cyanobacteria in Water a Guide to Their Public Health Consequences, Monitoring and Management, 2nd ed.; Chorus, I., Welker, M., Eds.; CRC Press: Boca Raton, FL, USA, 2021; pp. 72–94. [Google Scholar] [CrossRef]
  3. World Health Organization (WHO). Cyanobacterial Toxins: Anatoxin-a and Analogues; Background Document for Development of WHO Guidelines for Drinking-Water Quality and Guidelines for safe Recreational Water Environments; WHO: Geneva, Switzerland, 2020. [Google Scholar]
  4. Testai, E.; Scardala, S.; Vichi, S.; Buratti, F.M.; Funari, E. Risk to human health associated with the environmental occurrence of cyanobacterial neurotoxic alkaloids anatoxins and saxitoxins. Crit. Rev. Toxicol. 2016, 46, 385–419. [Google Scholar] [CrossRef]
  5. Ballot, A.; Krienitz, L.; Kotut, K.; Wiegand, C.; Pflugmacher, S. Cyanobacteria and cyanobacterial toxins in the alkaline crater lakes Sonachi and Simbi, Kenya. Harmful Algae 2005, 4, 139–150. [Google Scholar] [CrossRef]
  6. Kotut, K.; Ballot, A.; Krienitz, L. Toxic cyanobacteria and their toxins in standing waters of Kenya: Implications for water resource use. J. Water Health 2006, 4, 233–245. [Google Scholar] [CrossRef]
  7. Park, H.D.; Watanabe, M.F.; Harda, K.; Nagai, H.; Suzuki, M.; Watanabe, M.; Hayashi, H. Hepatotoxin (microcystin) and neurotoxin (anatoxin-a) contained in natural blooms and strains of cyanobacteria from Japanese freshwaters. Nat. Toxins 1993, 1, 353–360. [Google Scholar] [CrossRef]
  8. Joung, S.; Kim, J.H.; Ahn, C.; Choi, S.S.; Kim, H.S.; Oh, H.M. Water quality and cyanobacterial anatoxin-a concentration in Daechung reservoir. Korean J. Limnol. 2002, 35, 257–265. [Google Scholar]
  9. Carrasco, D.; Moreno, E.; Paniagua, T.; de Hoyos, C.; Wormer, L.; Sanchis, D.; Cirés, S.; Martín-del-Pozo, D.; Codd, G.; Quesada, A. Anatoxin-a occurrence and potential cyanobacterial anatoxin-a producers in Spanish reservoirs. J. Phycol. 2007, 43, 1120–1125. [Google Scholar] [CrossRef]
  10. Ballot, A.; Fastner, J.; Lentz, M.; Wiedner, C. First report of anatoxin-a-producing cyanobacterium Aphanizomenon issatschenkoi in northeastern Germany. Toxicon 2010, 56, 964–971. [Google Scholar] [CrossRef]
  11. Blahova, L.; Sehnal, L.; Lepsova-Skacelova, O.; Szmucova, V.; Babica, P.; Hilscherova, K.; Teikari, J.; Sivonen, K.; Blaha, L. Occurrence of cylindrospermopsin, anatoxin-a and their homologs in the southern Czech Republic—Taxonomical, analytical, and molecular approaches. Harmful Algae 2021, 108, 102101. [Google Scholar] [CrossRef] [PubMed]
  12. Bauer, F.; Wolfschlaeger, I.; Geist, J.; Fastner, J.; Schmalz, C.W.; Raeder, U. Occurrence, Distribution and Toxins of Benthic Cyanobacteria in German Lakes. Toxics 2023, 11, 643. [Google Scholar] [CrossRef] [PubMed]
  13. Ruiz, M.; Galanti, L.; Ruibal, A.L.; Rodríguez, M.I.; Wunderlin, D.A.; Amé, M.V. First report of microcystins and anatoxin-a co-occurrence in San Roque reservoir (Córdoba, Argentina). Water Air Soil Pollut. 2013, 224, 1593. [Google Scholar] [CrossRef]
  14. Bouma-Gregson, K.; Kudela, R.M.; Power, M.E. Widespread anatoxin-a detection in benthic cyanobacterial mats throughout a river network. PLoS ONE 2018, 13, e0197669. [Google Scholar] [CrossRef] [PubMed]
  15. McAllister, T.G.; Wood, S.A.; Atalah, J.; Hawes, I. Spatiotemporal dynamics of Phormidium cover and anatoxin concentrations in eight New Zealand rivers with contrasting nutrient and flow regimes. Sci. Total Environ. 2018, 612, 71–80. [Google Scholar] [CrossRef] [PubMed]
  16. John, N.; Baker, L.; Ansell, B.R.; Newham, S.; Crosbie, N.D.; Jex, A.R. First report of anatoxin-a producing cyanobacteria in Australia illustrates need to regularly up-date monitoring strategies in a shifting global distribution. Sci. Rep. 2019, 9, 10894. [Google Scholar] [CrossRef]
  17. Mazur, H.; Pliński, M. Nodularia spumigena blooms and the occurrence of hepatotoxin in the Gulf of Gdańsk. Oceanología 2003, 45, 305–316. [Google Scholar]
  18. Tango, P.J.; Butler, W. Cyanotoxins in Tidal Waters of Chesapeake Bay. Northeast. Nat. 2008, 15, 403–416. [Google Scholar] [CrossRef]
  19. Wood, S.A.; Rasmussen, J.P.; Holland, P.T.; Campbell, R.; Crowe, A.L.M. First report of the cyanotoxin anatoxin-a from Aphanizomenon issatschenkoi (cyanobacteria). J. Phycol. 2007, 43, 356–365. [Google Scholar] [CrossRef]
  20. Wood, S.A.; Smith, F.M.J.; Heath, M.W.; Palfroy, T.; Gaw, S.; Young, R.G.; Ryan, K.G. Within-mat variability in anatoxin-a and homoanatoxin-a production among benthic Phormidium (cyanobacteria) strains. Toxins 2012, 4, 900–912. [Google Scholar] [CrossRef]
  21. Gugger, M.; Lenoir, S.; Berger, C.; Ledreux, A.; Druart, J.; Humbert, J.; Guette, C.; Bernard, C. First report in a river in France of the benthic cyanobacterium Phormidium favosum producing anatoxin-a associated with dog neurotoxicosis. Toxicon 2005, 45, 919–928. [Google Scholar] [CrossRef] [PubMed]
  22. Fastner, J.; Beulker, C.; Geiser, B.; Hoffmann, A.; Kröger, R.; Teske, K.; Hoppe, J.; Mundhenk, L.; Neurath, H.; Sagebiel, D.; et al. Fatal neurotoxicosis in dogs associated with tychoplanktic, anatoxin-a producing Tychonema sp. in mesotrophic Lake Tegel, Berlin. Toxins 2018, 10, 60. [Google Scholar] [CrossRef]
  23. Bauer, F.; Fastner, J.; Bartha-Dima, B.; Breuer, W.; Falkenau, A.; Mayer, C.; Raeder, U. Mass ocurrence of anatoxin-a and dihydroanatoxin-a producing Tychonema sp. in mesotrophic reservoir mandichosee (River Lech, Germany) as a cause of neurotoxicosis in dogs. Toxins 2020, 12, 726. [Google Scholar] [CrossRef]
  24. Fastner, J.; Teikari, J.; Hoffmann, A.; Köhler, A.; Hoppe, S.; Dittmann, E.; Welker, M. Cyanotoxins associated with macrophytes in Berlin (Germany) water bodies—Occurrence and risk assessment. Sci. Total Environ. 2023, 858, 159433. [Google Scholar] [CrossRef] [PubMed]
  25. McCarron, P.; Rafuse, C.; Scott, S.; Lawrence, J.; Bruce, M.R.; Doothwright, E.; Murphy, C.; Reith, M.; Beach, D.G. Anatoxins from benthic cyanobacteria responsible for dog mortalities in New Brunswick, Canada. Toxicon 2023, 227, 107086. [Google Scholar] [CrossRef] [PubMed]
  26. Codd, G.A.; Testai, E.; Funari, E.; Sviřcev, Z. Cyanobacteria, Cyanotoxins, and Human Health. In Water Treatment for Purification from Cyanobacteria and Cyanotoxins, 1st ed.; Hiskia, E., Triantis, T.M., Antoniou, M.G., Kaloudis, T., Dionysiou, D.D., Eds.; John Wiley & Sons Ltd.: Chichester, UK, 2020; pp. 37–68. [Google Scholar]
  27. Biré, R.; Bertin, T.; Dom, I.; Hort, V.; Schmitt, C.; Diogène, J.; Lemée, R.; De Haro, L.; Nicolas, M. First Evidence of the presence of anatoxin-a in sea figs associated with human food poisonings in France. Mar. Drugs 2020, 18, 285. [Google Scholar] [CrossRef]
  28. Amzil, Z.; Derrien, A.; Terre Terrillon, A.; Savar, V.; Bertin, T.; Peyrat, M.; Duval, A.; Lhaute, K.; Arnich, N.; Hort, V.; et al. Five years monitoring the emergence of unregulated toxins in shellfish in France (EMERGTOX 2018-2022). Mar. Drugs 2023, 21, 435. [Google Scholar] [CrossRef] [PubMed]
  29. Swanson, K.L.; Allen, C.N.; Aronstam, R.S.; Rapoport, H.; Albuquerque, E.X. Molecular mechanisms of the potent and stereospecific nicotinic receptor agonist (+)-anatoxin-a. Mol. Pharmacol. 1986, 29, 250–257. [Google Scholar]
  30. Zhang, X.; Stjernlöf, P.; Adem, A.; Nordberg, A. Anatoxin-a a potent ligand for nicotinic cholinergic receptors in rat brain. Eur. J. Pharmacol. 1987, 135, 457–458. [Google Scholar] [CrossRef]
  31. Molloy, L.; Wonnacott, S.; Gallagher, T.; Brough, P.A.; Livett, B.G. Anatoxin-a is a potent agonist of the nicotinic acetylcholine receptor of bovine adrenal chromaffin cells. Eur. J. Pharmacol. 1995, 289, 447–453. [Google Scholar] [CrossRef] [PubMed]
  32. Takser, L.; Benachour, N.; Husk, B.; Cabana, H.; Gris, D. Cyanotoxins at low doses induce apoptosis and inflammatory effects in murine brain cells: Potential implications for neurodegenerative diseases. Toxicol. Rep. 2016, 3, 180–189. [Google Scholar] [CrossRef] [PubMed]
  33. Kubickova, B.; Martinkova, S.; Bohaciakova, D.; Hilscherova, K. Cyanobacterial anatoxin-a does not induce in vitro developmental neurotoxicity, but changes gene expression patterns in co-exposure with all-trans retinoic acid. Toxicol. Lett. 2024, 391, 39–44. [Google Scholar] [CrossRef]
  34. Rao Lakshmana, P.V.; Bhattacharya, R.; Gupta, N.; Parida, M.M.; Bhaskar, A.S.; Dubey, R. Involvement of caspase and reactive oxygen species in cyanobacterial toxin anatoxin-a-induced cytotoxicity and apoptosis in rat thymocytes and Vero cells. Arch. Toxicol. 2002, 76, 227–235. [Google Scholar] [CrossRef]
  35. Teneva, I.; Mladenov, R.; Popov, N.; Dzhambazov, B. Cytotoxicity and apoptotic effects of microcystin-LR and anatoxin-a in mouse lymphocytes. Folia Biol. 2005, 51, 62–67. [Google Scholar]
  36. Bownik, A.; Rymuszka, A.; Sierosławska, A.; Skowronski, T. Anatoxin-a induces apoptosis of leukocytes and decreases the proliferative ability of lymphocytes of common carp (Cyprinus carpio L.) in vitro. Pol. J. Vet. Sci. 2012, 15, 531–535. [Google Scholar] [CrossRef]
  37. Zhong, Y.; Shen, L.; Ye, X.; Zhou, D.; He, Y.; Li, Y.; Ding, Y.; Zhu, W.; Ding, J.; Zhang, H. Neurotoxic anatoxin-a can also exert immunotoxicity by the induction of apoptosis on Carassius auratus lymphocytes in vitro when exposed to environmentally relevant concentrations. Front. Physiol. 2020, 11, 316. [Google Scholar] [CrossRef]
  38. Rymuszka, A. Cytotoxic activity of the neurotoxin anatoxin-a on fish leukocytes in vitro and in vivo studies. Acta Vet. Brno 2012, 81, 175–182. [Google Scholar] [CrossRef]
  39. Adamski, M.; Zimolag, E.; Kaminski, A.; Drukała, J.; Bialczyk, J. Effects of cylindrospermopsin, its decomposition products, and anatoxin-a on human keratinocytes. Sci. Total Environ. 2021, 765, 142670. [Google Scholar] [CrossRef] [PubMed]
  40. Plata-Calzado, C.; Diez-Quijada, L.; Medrano-Padial, C.; Prieto, A.I.; Cameán, A.M.; Jos, A. In vitro mutagenic and genotoxic assessment of anatoxin-a alone and in combination with cylindrospermopsin. Toxins 2023, 15, 458. [Google Scholar] [CrossRef] [PubMed]
  41. Sirén, A.L.; Feuerstein, G. Cardiovascular effects of anatoxin-A in the conscious rat. Toxicol. Appl. Pharmacol. 1990, 102, 91–100. [Google Scholar] [CrossRef] [PubMed]
  42. Adeyemo, O.M.; Sirén, A.L. Cardio-respiratory changes and mortality in the conscious rat induced by (+)- and (±)-anatoxin-a. Toxicon 1992, 30, 899–905. [Google Scholar] [CrossRef]
  43. Oberemm, A.; Becker, J.; Codd, G.A.; Steinberg, C. Effects of cyanobacterial toxins and aqueous crude extracts of cyanobacteria on the development of fish and amphibians. Environ. Toxicol. Int. J. 1999, 14, 77–88. [Google Scholar] [CrossRef]
  44. Abdallah, M.; Van Hassel, W.H.R.; Andjelkovic, M.; Wilmotte, A.; Rajkovic, A. Cyanotoxins and food contamination in developing countries: Review of their types, toxicity, analysis, occurrence and mitigation strategies. Toxins 2021, 13, 786. [Google Scholar] [CrossRef]
  45. Pichardo, S.; Cameán, A.M.; Jos, A. In Vitro Toxicological Assessment of Cylindrospermopsin: A Review. Toxins 2017, 9, 402. [Google Scholar] [CrossRef]
  46. Plata-Calzado, C.; Prieto, A.I.; Cameán, A.M.; Jos, A. Toxic Effects Produced by Anatoxin-a under Laboratory Conditions: A Review. Toxins 2022, 14, 861. [Google Scholar] [CrossRef]
  47. Lukas, R.J.; Norman, S.A.; Lucero, L. Characterization of nicotinic acetylcholine receptors expressed by cells of the SH-SY5Y human neuroblastoma clonal line. Mol. Cell Neurosci. 1993, 4, 1–12. [Google Scholar] [CrossRef] [PubMed]
  48. Valero, T.; Jacobs, T.; Moschopoulou, G.; Naumann, M.; Hauptmann, P.; Kintzios, S. Electrical impedance analysis of N2a neuroblastoma cells in gel matrices after ACh-receptor triggering with an impedimetric biosensor. Procedia Chem. 2009, 1, 734–737. [Google Scholar] [CrossRef]
  49. Hinojosa, M.G.; Prieto, A.I.; Gutiérrez-Praena, D.; Moreno, F.J.; Cameán, A.M.; Jos, A. Neurotoxic assessment of Microcystin-LR, cylindrospermopsin and their combination on the human neuroblastoma SH-SY5Y cell line. Chemosphere 2019, 224, 751–764. [Google Scholar] [CrossRef]
  50. Osswald, J.; Carvalho, A.P.; Guimarães, L.; Guilhermino, L. Toxic effects of pure anatoxin-a on biomarkers on rainbow trout, Oncorhynchus mykiss. Toxicon 2013, 70, 162–169. [Google Scholar] [CrossRef] [PubMed]
  51. Liu, J.; Hernández, S.E.; Swift, S.; Singhal, N. Estrogenic activity of cylindrospermopsin and anatoxin-a and their oxidative products by FeIII-B*/H2O2. Water Res. 2018, 132, 309–319. [Google Scholar] [CrossRef] [PubMed]
  52. Diez-Quijada, L.; Puerto, M.; Gutiérrez-Praena, D.; Turkina, M.V.; Campos, A.; Vasconcelos, V.; Cameán, A.M.; Jos, A. In Vitro toxicity evaluation of cyanotoxins cylindrospermopsin and microcystin-LR on human kidney HEK293 cells. Toxins 2022, 14, 429. [Google Scholar] [CrossRef] [PubMed]
  53. Casas-Rodríguez, A.; Cebadero-Dominguez, Ó.; Puerto, M.; Cameán, A.M.; Jos, A. Immunomodulatory effects of cylindrospermopsin in human T cells and monocytes. Toxins 2023, 15, 301. [Google Scholar] [CrossRef] [PubMed]
  54. Menezes, C.; Valerio, E.; Días, E. The kidney vero-E6 cell line: A suitable model to study the toxicity of microcystins. In New Insights into Toxicity and Drug Testing; Gowder, S., Ed.; IntechOpen: London, UK, 2013. [Google Scholar] [CrossRef]
  55. Gutiérrez-Praena, D.; Pichardo, S.; Jos, A.; Moreno, F.J.; Cameán, A.M. Biochemical and pathological toxic effects induced by the cyanotoxin Cylindrospermopsin on the human cell line Caco-2. Water Res. 2012, 46, 1566–1575. [Google Scholar] [CrossRef] [PubMed]
  56. Gutiérrez-Praena, D.; Guzmán-Guillén, R.; Pichardo, S.; Moreno, F.J.; Vasconcelos, V.; Jos, A.; Cameán, A.M. Cytotoxic and morphological effects of microcystin-LR, cylindrospermopsin, and their combinations on the human hepatic cell line HepG2. Environ. Toxicol. 2018, 34, 240–251. [Google Scholar] [CrossRef] [PubMed]
  57. World Health Organization (WHO). Cyanobacterial Toxins: Cylindrospermopsins; Background Document for Development of WHO Guidelines for Drinking-Water Quality and Guidelines for Safe Recreational Water Environments; WHO: Geneva, Switzerland, 2020. [Google Scholar]
  58. Dimitrikapoulos, I.K.; Kaloudis, T.S.; Hiskia, A.E.; Thomaidis, N.S.; Koupparis, M. Development of a fast and selective method for the sensitive determination of anatoxin-a in lake waters using liquid chromatography–tandem mass spectrometry and phenylalanine-d5 as internal standard. Anal. Bioanal. Chem. 2010, 397, 2245–2252. [Google Scholar] [CrossRef] [PubMed]
  59. Plata-Calzado, C.; Prieto, A.I.; Cameán, A.M.; Jos, A. Analytical methods for anatoxin-a determination: A review. Toxins 2024, 16, 198. [Google Scholar] [CrossRef] [PubMed]
  60. Guzmán-Guillén, R.; Prieto, A.I.; Moreno, I.; González, G.; Soria-Díaz, M.E.; Vasconcelos, V.; Cameán, A.M. Development and optimization of a method for the determination of Cylindrospermopsin from strains of Aphanizomenon cultures: Intra-laboratory assessment of its accuracy by using validation standards. Talanta 2012, 100, 356–363. [Google Scholar] [CrossRef]
  61. Osswald, J.; Rellán, S.; Gago, A.; Vasconcelos, V. Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, anatoxin-a. Environ. Int. 2007, 33, 1070–1089. [Google Scholar] [CrossRef] [PubMed]
  62. Christensen, V.G.; Khan, E. Freshwater neurotoxins and concerns for human, animal, and ecosystem health: A review of anatoxin-a and saxitoxin. Sci. Total Environ. 2020, 736, 19515. [Google Scholar] [CrossRef] [PubMed]
Toxins 16 00541 g001
Figure 1. Reduction in tetrazolium salt MTS in (a) neuronal cells (SH-SY5Y and N2a), (b) Caco-2 and HepG2 cells and (c) immune system cells (THP-1, Jurkat and L5178Y Tk+/−) after 24 h of exposure to 0–200 µg/mL ATX-a fumarate. In addition, 0.3% v/v Triton X-100 was used as the positive control. All experiments were performed at least in triplicate per concentration. Data are expressed as mean ± SD compared to negative control group. Note: * p < 0.05 and *** p < 0.001 indicated significant difference from negative control.
Figure 1. Reduction in tetrazolium salt MTS in (a) neuronal cells (SH-SY5Y and N2a), (b) Caco-2 and HepG2 cells and (c) immune system cells (THP-1, Jurkat and L5178Y Tk+/−) after 24 h of exposure to 0–200 µg/mL ATX-a fumarate. In addition, 0.3% v/v Triton X-100 was used as the positive control. All experiments were performed at least in triplicate per concentration. Data are expressed as mean ± SD compared to negative control group. Note: * p < 0.05 and *** p < 0.001 indicated significant difference from negative control.
Toxins 16 00541 g001
Toxins 16 00541 g002
Figure 2. Reduction in tetrazolium salt MTS in THP-1 cells exposed to 0–3 µg/mL CYN for 24 h. In addition, 0.3% v/v Triton X-100 was used as the positive control. Experiments were performed in triplicate per concentration. Data are expressed as mean ± SD compared to negative control group. Note: * p < 0.05 and *** p < 0.001 indicate significant difference from the negative control.
Figure 2. Reduction in tetrazolium salt MTS in THP-1 cells exposed to 0–3 µg/mL CYN for 24 h. In addition, 0.3% v/v Triton X-100 was used as the positive control. Experiments were performed in triplicate per concentration. Data are expressed as mean ± SD compared to negative control group. Note: * p < 0.05 and *** p < 0.001 indicate significant difference from the negative control.
Toxins 16 00541 g002
Toxins 16 00541 g003
Figure 3. Chromatograms obtained by UHPLC-MS/MS of ATX-a in THP-1 cells exposed to 50 µg/mL ATX-a fumarate for 24 h. (a) Negative control, (b) intracellular fraction and (c) extracellular fraction.
Figure 3. Chromatograms obtained by UHPLC-MS/MS of ATX-a in THP-1 cells exposed to 50 µg/mL ATX-a fumarate for 24 h. (a) Negative control, (b) intracellular fraction and (c) extracellular fraction.
Toxins 16 00541 g003
Toxins 16 00541 g004
Figure 4. Effects of ATX-a on the expression of mRNA of genes involved in apoptosis/necrosis in THP-1 cells. Cells were exposed to 5 or 50 µg/mL ATX-a fumarate for 24 h. CPT (0.5 µM) was used as positive control and chloroform/methanol (4:1) as solvent control. Results are expressed as relative mRNA expression normalized to the negative control group. * p < 0.05 and **** p < 0.0001 indicate significantly difference from negative controls.
Figure 4. Effects of ATX-a on the expression of mRNA of genes involved in apoptosis/necrosis in THP-1 cells. Cells were exposed to 5 or 50 µg/mL ATX-a fumarate for 24 h. CPT (0.5 µM) was used as positive control and chloroform/methanol (4:1) as solvent control. Results are expressed as relative mRNA expression normalized to the negative control group. * p < 0.05 and **** p < 0.0001 indicate significantly difference from negative controls.
Toxins 16 00541 g004
Table 1. Mean ± SD (%) of intracellular and extracellular ATX-a in N2a, SH-SY5Y, L5178YTk+/−, Jurkat, THP-1, HepG2, and Caco-2 cell lines after exposure to 50 or 100 µg/mL ATX-a fumarate for a period of 24 h by triplicate. n.d.: not detected.
Table 1. Mean ± SD (%) of intracellular and extracellular ATX-a in N2a, SH-SY5Y, L5178YTk+/−, Jurkat, THP-1, HepG2, and Caco-2 cell lines after exposure to 50 or 100 µg/mL ATX-a fumarate for a period of 24 h by triplicate. n.d.: not detected.
ATX-a Fumarate (µg/mL)% Intracellular
Mean ± SD		% Extracellular
Mean ± SD		% Total ATX-a Detected
Mean ± SD
N2aC−n.d.n.d.n.d.
50 µg/mL1.71 ± 1.1559.90 ± 4.3261.61 ± 3.59
100 µg/mL1.52 ± 0.6642.03 ± 11.7343.55 ± 11.36
SH-SY5YC−n.d.n.d.n.d.
50 µg/mL1.99 ± 0.67127.85 ± 8.71129.84 ± 9.38
100 µg/mL2.94 ± 1.91106.91 ± 39.49109.85 ± 40.62
L5178YTk+/−C−n.d.n.d.n.d.
50 µg/mL9.70 ± 4.7872.61 ± 9.5482.31 ± 10.34
100 µg/mL6.45 ± 1.3169.44 ± 10.5075.89 ± 9.45
JurkatC−n.d.n.d.n.d.
50 µg/mL10.58 ± 0.7475.21 ± 23.2385.78 ± 23.85
100 µg/mL13.96 ± 3.4874.14 ± 15.5888.11 ± 15.99
THP-1C−n.d.n.d.n.d.
50 µg/mL14.72 ± 4.5593.90 ± 3.35108.62 ± 7.46
100 µg/mL6.62 ± 1.3756.35 ± 5.3562.98 ± 4.80
HepG2C−n.d.n.d.n.d.
50 µg/mL0.65 ± 0.05104.03 ± 2.82104.72 ± 2.87
100 µg/mL0.30 ± 0.0262.52 ± 4.8762.81 ± 4.89
Caco-2C−n.d.n.d.n.d.
50 µg/mL0.46 ± 0.1583.72 ± 30.8584.18 ± 30.75
100 µg/mL0.52 ± 0.2272.02 ± 21.8872.54 ± 21.88
Table 2. Mean ± SD (%) of intracellular and extracellular CYN in THP-1 cells after exposure to 0. 1.1, 2.5 and 3 µg/mL CYN for a period of 24 h by triplicate. n.d.: not detected.
Table 2. Mean ± SD (%) of intracellular and extracellular CYN in THP-1 cells after exposure to 0. 1.1, 2.5 and 3 µg/mL CYN for a period of 24 h by triplicate. n.d.: not detected.
CYN (µg/mL)% Intracellular
Mean ± SD		% Extracellular
Mean ± SD		% Total CYN Detected
Mean ± SD
THP-1C−n.d.n.d.n.d.
1.1 µg/mL0.38 ± 0.0559.74 ± 1.0160.12 ± 1.06
2.5 µg/mL0.71 ± 0.1657.10 ± 2.1957.81 ± 2.21
3 µg/mL1.30 ± 0.5864.56 ± 4.2965.85 ± 4.83
Table 3. Cell lines employed in the study and culture conditions for the maintenance.
Table 3. Cell lines employed in the study and culture conditions for the maintenance.
Cell LineCell TypeTissue TypeModel
Organism		Culture MediaCulture
Conditions
N2aNeuroblastBrainMouse43.5% OptiMEM + 43.5% DMEM high glucose + 10% FBS, 1% piruvate 1% L-glutamine and 1% penicillin–streptomycinIncubator at 37 °C with 95% relative humidity and 5% CO2
SH-SY5YNeuroblastBrainHuman43% MEM + 43% Ham’s F12 + 10% FBS, 1% piruvate 1% L-glutamine, 1% NEAA and 1% penicillin–streptomycin
JurkatT lymphoblastPeripheral bloodHumanRPMI 1640 medium containing high glucose (R8005, Sigma Aldrich) + 10% FBS, 1% penicillin–streptomycin and 2g/L sodium bicarbonate
THP-1MonocytePeripheral bloodHuman
L5178Y Tk+/−LymphoblastLymph nodeMouseRPMI 1640 medium + 10% horse serum, 1% pyruvate, 1% L-glutamine and 1% penicillin–streptomycin
Caco-2Epithelial cellColonHumanMEM medium + 10% FBS, 1% NEAA, 1% pyruvate, 1% L-glutamine and 1% penicillin–streptomycin
HepG2HepatocyteLiverHumanMEM medium + 10% FBS, 1% L-glutamine and 1% penicillin–streptomycin
DMEM: Dulbecco’s modified eagle; MEM: minimum essential medium FBS: fetal bovine serum; RPMI: Roswell Park Memorial Institute; NEAA: non-essential amino acids.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Plata-Calzado, C.; Prieto, A.I.; Cameán, A.M.; Jos, A. Assessment of the Effects of Anatoxin-a In Vitro: Cytotoxicity and Uptake. Toxins 2024, 16, 541. https://doi.org/10.3390/toxins16120541

AMA Style

Plata-Calzado C, Prieto AI, Cameán AM, Jos A. Assessment of the Effects of Anatoxin-a In Vitro: Cytotoxicity and Uptake. Toxins. 2024; 16(12):541. https://doi.org/10.3390/toxins16120541

Chicago/Turabian Style

Plata-Calzado, Cristina, Ana I. Prieto, Ana M. Cameán, and Angeles Jos. 2024. "Assessment of the Effects of Anatoxin-a In Vitro: Cytotoxicity and Uptake" Toxins 16, no. 12: 541. https://doi.org/10.3390/toxins16120541

APA Style

Plata-Calzado, C., Prieto, A. I., Cameán, A. M., & Jos, A. (2024). Assessment of the Effects of Anatoxin-a In Vitro: Cytotoxicity and Uptake. Toxins, 16(12), 541. https://doi.org/10.3390/toxins16120541

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop