Characterization of NR1J1 Paralog Responses of Marine Mussels: Insights from Toxins and Natural Activators

Citation: Casas-Rodríguez, A.; Medrano-Padial, C.; Jos, A.; Cameán, A.M.; Campos, A.; Fonseca, E. Characterization of NR1J1 Paralog Responses of Marine Mussels: Insights from Toxins and Natural Activators. Int. J. Mol. Sci. 2024,25, 6287. https://doi.org/10.3390/ ijms25126287 Academic Editor: Se-Kwon Kim Received: 6 May 2024 Revised: 30 May 2024 Accepted: 30 May 2024 Published: 7 June 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Characterization of NR1J1 Paralog Responses of Marine Mussels: Insights from Toxins and Natural Activators Antonio Casas-Rodríguez 1,†, Concepción Medrano-Padial 1,2,*,† , Angeles Jos 1, Ana M. Cameán1, Alexandre Campos 3,* and Elza Fonseca 3 1Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n◦2, 41012 Seville, Spain; [email protected] (A.C.-R.); [email protected] (A.J.); [email protected] (A.M.C.) 2Laboratorio de Fitoquímica y Alimentos Saludables (LabFAS), Centro de Edafología y Biología Aplicada del Segura, Consejo Superior de Investigaciones Científicas (CEBAS-CSIC), Campus Universitario 25, Espinardo, 30100 Murcia, Spain 3Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, s/n, 4450-208 Matosinhos, Portugal; [email protected] *Correspondence: [email protected] (C.M.-P.); [email protected] (A.C.) †These authors contributed equally to this work. Abstract: The pregnane X receptor (PXR) is a nuclear hormone receptor that plays a pivotal role in regulating gene expression in response to various ligands, particularly xenobiotics. In this context, the aim of this study was to shed light on the ligand affinity and functions of four NR1J1 paralogs identified in the marine mussel Mytilus galloprovincialis, employing a dual-luciferase reporter assay. To achieve this, the activation patterns of these paralogs in response to various toxins, including freshwater cyanotoxins (Anatoxin-a, Cylindrospermopsin, and Microcystin-LR, -RR, and -YR) and marine algal toxins (Nodularin, Saxitoxin, and Tetrodotoxin), alongside natural compounds (Saint John’s Wort, Ursolic Acid, and 8-Methoxypsoralene) and microalgal extracts (Tetraselmis,Isochrysis, LEGE 95046, and LEGE 91351 extracts), were studied. The investigation revealed nuanced differences in paralog response patterns, highlighting the remarkable sensitivity of MgaNR1J1 γ and MgaNR1J1 δ paralogs to several toxins. In conclusion, this study sheds light on the intricate mechanisms of xenobiotic metabolism and detoxification, particularly focusing on the role of marine mussel NR1J1 in responding to a diverse array of compounds. Furthermore, comparative analysis with human PXR revealed potential species-specific adaptations in detoxification mechanisms, suggesting evolutionary implications. These findings deepen our understanding of PXR-mediated metabolism mechanisms, offering insights into environmental monitoring and evolutionary biology research. Keywords: pregnane X receptor; NR1J1 paralogs; bivalves; cyanotoxins; toxins; microalgal extracts; marine mussels 1. Introduction Nuclear hormone receptors (NRs) are transcription factors associated with co-factors that regulate gene expression, usually upon binding to a ligand [ 1 ]. They are proteins exclusive to metazoans and expressed in different tissues [ 2 ]. Seven subfamilies have been identified in mammals, each performing varied molecular and physiological functions, encompassing gene regulation and intracellular metabolic and physiological homeostasis, as well as orchestrating cellular differentiation and developmental processes [ 3 – 5 ]. The current hypothesis suggests that NRs emerged early in animal evolution from ancestral receptors that first appeared in invertebrates evolving through a series of gene amplifications and subsequent mutations and diversifications [ 6 , 7 ]. This theory is consistent with the absence of several NR orthologous genes in invertebrates, such as some vertebrate endocrine hormone targets (e.g., androgen receptor AR, glucocorticoid receptor GR) from subfamily Int. J. Mol. Sci. 2024,25, 6287. https://doi.org/10.3390/ijms25126287 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2024,25, 6287 2 of 16 3 group C [ 8 ]. NRs are part of the endocrine system regulating and coordinating multiple processes involved in lipid and cholesterol metabolism and bile salt synthesis [ 8 ], in addition to playing a critical role in embryonic and post-embryonic development [ 9 , 10 ]. Hence, the impairment of NR signaling has been related to several metabolic and inflammatory diseases [3,11]. Most NRs share a conserved modular structure that includes, from the N-terminus to the C-terminus, the modulatory A/B domain, the DNA-binding domain (DBD, C domain), the ‘hinge’ D domain, the ligand-binding domain (LBD, E domain), and a variable Cterminal F domain that can be absent in some NRs [ 6 , 12 ]. The DBD mediates NR binding to DNA sequence-specific response elements. DBDs are highly conserved among species, comprising about 80 amino acids that fold to form two zinc fingers, each composed of four cysteine residues that chelate a zinc atom. In turn, LBDs mediate ligand recognition and binding, consisting of approximately 250 amino acids that fold into a hydrophobic pocket where ligands bind [12]. The pregnane X receptor (PXR) belongs to subfamily 1 group I (NR1I2) [ 13 ] and is considered a key element in the defense against toxic substances, including foreign chemicals (xenobiotics) [ 14 ]. The name ‘pregnane’ X receptor came from its activation by pregnane (21-carbon or C21) steroids such as progesterone or 5 β -pregnan-3,20-dione [ 14 ]. PXR exerts a significant influence on metabolism modulation, cell cycle arrest, inflammation, and angiogenesis [ 15 ]. Moreover, it regulates and coordinates xenobiotic metabolism, which includes oxidation and conjugation reactions and the transport of metabolites (xenobiotics) [ 12 ]. Given these functions, PXR is a primary xenobiotic sensor with a critical role in protecting against chemical challenges. Compared to other NRs, PXR modulates a broad spectrum of biological processes, and the differences among orthologous sequences are consistent with the extraordinary differences in PXR ligand specificities across vertebrate species, supporting the theory that PXR evolved to adapt to cross-species differences in exogenous and endogenous toxic compounds [ 14 , 16 ]. Among the endogenous and exogenous chemicals that activate vertebrate PXR are steroids, bile acids, environmental pollutants, and prescription drugs [ 12 ]. Mammalian and vertebrate PXRs regulate genes from phases I (cyp3A family members, cyp2B6, cyp2b9, cyp2C8, cyp2C9, and cyp2C19), II (glutathione-Stransferase, sulfotransferase, UDP-glucuronosyltransferase, and carboxylesterase family genes), and III (hepatic transporters oatp2, mrp2, and mdr1) of xenobiotic detoxification [ 12 ]. Although vertebrate PXR orthologs have been extensively characterized, our understating of this gene in invertebrates, particularly aquatic species, remains limited. The diversity of NR complements across various marine invertebrate phyla is remarkable. These variations might be linked to the wide array of life cycles, developmental strategies, and reproductive adaptations observed among marine invertebrates [ 17 ]. The current evolutionary hypothesis regarding NR1I and NR1J homologs suggests that this gene lineage diverged early in the protostome–deuterostome split [ 18 ]. An open reading frame encoding a VDR/PXR/CAR-like ortholog (NR1J1 β ) from the estuarine bivalve peppery furrow shell (Scrobicularia plana) was recently isolated and characterized. In this study, NR1J1 was suggested to participate in the detoxification mechanisms of mollusks as its activity was modulated in the presence of the natural toxin okadaic acid (OA) and pesticides in the nanomolar range [18]. Bivalve mollusks constitute an important ecological group of aquatic filter-feeders [ 19 ]. Furthermore, filter-feeding behavior is associated with the exposure to and accumulation of a wide variety of natural and human-made chemicals that are potentially harmful to animals, including microalgal biotoxins [ 20 ]. Given the anthropogenic pressures they face, it is imperative to safeguard aquatic systems and their communities, especially bivalve populations, and enhance water quality through pollution reduction efforts [ 21 ]. It should be considered as a possibility that this unique mode of living and habitat conditions have driven the adaptive evolution of NR1J1 for bivalves to sense and cope with the multiple chemical challenges that come from the environment. This work aims to shed light on the ligand affinity and functions of the four NR1J1 paralogs identified in the marine mussel Int. J. Mol. Sci. 2024,25, 6287 3 of 16 Mytilus galloprovincialis, employing a dual-luciferase reporter assay. Among the substances tested as putative NR1J1 ligand activators were several microalgal toxins and microalgal extracts known to constitute potential chemical challenges in this species. 2. Results The dual-luciferase reporter gene assay is a method used to evaluate gene expression regulation. The results illustrate the luciferase reporter gene expression levels in terms of the luciferase activity fold change following exposure to freshwater cyanotoxins (Figures 1and 2) , marine toxins (Figure 3), non-toxic natural compounds (Figure 4), and algal extracts (Figure 5). Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 of 16 conditions have driven the adaptive evolution of NR1J1 for bivalves to sense and cope with the multiple chemical challenges that come from the environment. This work aims to shed light on the ligand affinity and functions of the four NR1J1 paralogs identified in the marine mussel Mytilus galloprovincialis, employing a dual-luciferase reporter assay. Among the substances tested as putative NR1J1 ligand activators were several microalgal toxins and microalgal extracts known to constitute potential chemical challenges in this species. 2. Results The dual-luciferase reporter gene assay is a method used to evaluate gene expression regulation. The results illustrate the luciferase reporter gene expression levels in terms of the luciferase activity fold change following exposure to freshwater cyanotoxins (Figures 1 and 2), marine toxins (Figure 3), non-toxic natural compounds (Figure 4), and algal extracts (Figure 5). Figure 1. Firefly luciferase transactivation activity mediated by marine mussel NR1J1 paralogs with the freshwater cyanotoxins (A) Anatoxin-A and (B) Cylindrospermopsin. Human PXR was used as a control assay and OA as a positive control. Values are expressed as mean ± SEM of three replicates. Distinct lowercase letters indicate values significantly different at p < 0.01 according to one-way analysis of variance (ANOVA) per gene and Tukey’s multiple range test (n = 3). Figure 1. Firefly luciferase transactivation activity mediated by marine mussel NR1J1 paralogs with the freshwater cyanotoxins (A) Anatoxin-A and (B) Cylindrospermopsin. Human PXR was used as a control assay and OA as a positive control. Values are expressed as mean ± SEM of three replicates. Distinct lowercase letters indicate values significantly different at p< 0.01 according to one-way analysis of variance (ANOVA) per gene and Tukey’s multiple range test (n= 3). Int. J. Mol. Sci. 2024,25, 6287 4 of 16 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 of 16 Figure 2. Firefly luciferase transactivation activity mediated by marine mussel NR1J1 paralogs with the freshwater cyanotoxins (A) Microcystin-LR, (B) Microcystin-RR, and (C) Microcystin-YR. Human PXR was used as a control assay and OA as a positive control. Values are expressed as mean ± SEM of three replicates. Distinct lowercase letters indicate values significantly different at p < 0.01 according to one-way analysis of variance (ANOVA) per gene and Tukey’s multiple range test (n = 3). Figure 2. Firefly luciferase transactivation activity mediated by marine mussel NR1J1 paralogs with the freshwater cyanotoxins (A) Microcystin-LR, (B) Microcystin-RR, and (C) Microcystin-YR. Human PXR was used as a control assay and OA as a positive control. Values are expressed as mean ±SEM of three replicates. Distinct lowercase letters indicate values significantly different at p< 0.01 according to one-way analysis of variance (ANOVA) per gene and Tukey’s multiple range test (n= 3). Int. J. Mol. Sci. 2024,25, 6287 5 of 16 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 5 of 16 Figure 3. Firefly luciferase transactivation activity mediated by marine mussel NR1J1 paralogs with 3 marine algal toxins: (A) Nodularin, (B) Saxitoxin, and (C) Tetrodotoxin. Human PXR was used as a control assay and OA as a positive control. Values are expressed as mean ± SEM of three replicates. Distinct lowercase letters indicate values significantly different at p < 0.01 according to one-way analysis of variance (ANOVA) per gene and Tukey’s multiple range test (n = 3). Figure 3. Firefly luciferase transactivation activity mediated by marine mussel NR1J1 paralogs with 3 marine algal toxins: (A) Nodularin, (B) Saxitoxin, and (C) Tetrodotoxin. Human PXR was used as a control assay and OA as a positive control. Values are expressed as mean ± SEM of three replicates. Distinct lowercase letters indicate values significantly different at p< 0.01 according to one-way analysis of variance (ANOVA) per gene and Tukey’s multiple range test (n= 3). Int. J. Mol. Sci. 2024,25, 6287 6 of 16 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6 of 16 Figure 4. Firefly luciferase transactivation activity mediated by marine mussel NR1J1 paralogs with 3 natural non-toxic compounds: (A) Saint John’s Wort, (B) Ursolic Acid, and (C) 8-Methoxypsoralene. Human PXR was used as a control assay and OA as a positive control. Values are expressed as mean ± SEM of three replicates. Distinct lowercase letters indicate values significantly different at p < 0.01 according to one-way analysis of variance (ANOVA) per gene and Tukey’s multiple range test (n = 3). Figure 4. Firefly luciferase transactivation activity mediated by marine mussel NR1J1 paralogs with 3 natural non-toxic compounds: (A) Saint John’s Wort, (B) Ursolic Acid, and (C) 8-Methoxypsoralene. Human PXR was used as a control assay and OA as a positive control. Values are expressed as mean ±SEM of three replicates. Distinct lowercase letters indicate values significantly different at p< 0.01 according to one-way analysis of variance (ANOVA) per gene and Tukey’s multiple range test (n= 3). Int. J. Mol. Sci. 2024,25, 6287 7 of 16 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 of 16 Figure 5. Firefly luciferase transactivation activity mediated by marine mussel NR1J1 α paralog with 4 microalgae extracts: (A) Tetraselmis, (B) Isochrysis, (C) LEGE CC 95046, and (D) LEGE CC 91351. Human PXR was used as a control assay and OA as a positive control. Values are expressed as mean ± SEM of three replicates. Distinct lowercase letters indicate values significantly different at p < 0.01 according to one-way analysis of variance (ANOVA) per gene and Tukey’s multiple range test (n = 3). 2.1. Freshwater Cyanotoxins In the presence of both Anatoxin-A (ATX-A) (Figure 1A) and Cylindrospermopsin (CYN) (Figure 1B), an activation of the human PXR (HsaPXR) and marine mussel NR1J1 (MgaNR1J1) receptors was observed. Significant differences were observed in all paralogs at 100 nM ATX-A compared to the negative control. Exposure to 100 nM ATX-A resulted in approximately 9and 6-fold increases in the luciferase signals of MgaNR1J1γ and MgaNR1J1δ, respectively. In contrast, the induction in the other paralogs studied was lower, reaching up to 4-fold. Notably, no significant differences were observed between the concentrations tested in the human homolog (Figure 1A). Similarly, regarding CYN exposure (Figure 1B), the most sensitive paralogs were MgaNR1J1γ and MgaNR1J1δ. At the highest concentration of CYN tested (100 nM), the signal intensity of these paralogs was comparable to that induced by OA. MgaNR1J1δ exhibited the maximum activity, reaching an approximately 6-fold increase compared to the negative control. In contrast, HsaPXR was the least sensitive paralog. The data obtained from different congeners of Microcystins (MCs) are represented in Figure 2. Overall, MgaNR1J1 paralogs were significantly transactivated in a concentration-dependent manner, while HsaPXR did not respond differently to the concentrations of MCs assayed. Although the results obtained with MC-LR were significantly different, it caused a smaller effect among the congeners studied (Figure 2A). A statistical analysis indicated that the most sensitive paralogs were MgaNR1J1γ and MgaNR1J1δ. Moreover, there were no discernible differences between concentrations except for MgaNR1J1α. Similar trends were obtained when transfected COS-1 cells were exposed to MC-RR (Figure 2B) and MC-YR (Figure 2C). The MgaNR1J1γ paralog was the most sensitive to the exposure to both toxins at 100 nM, exhibiting an increase of up to 9-fold in its activity. Significant differences between the two tested Figure 5. Firefly luciferase transactivation activity mediated by marine mussel NR1J1 α paralog with 4 microalgae extracts: (A)Tetraselmis, (B)Isochrysis, (C) LEGE CC 95046, and (D) LEGE CC 91351. Human PXR was used as a control assay and OA as a positive control. Values are expressed as mean ±SEM of three replicates. Distinct lowercase letters indicate values significantly different at p< 0.01 according to one-way analysis of variance (ANOVA) per gene and Tukey’s multiple range test (n= 3). 2.1. Freshwater Cyanotoxins In the presence of both Anatoxin-A (ATX-A) (Figure 1A) and Cylindrospermopsin (CYN) (Figure 1B), an activation of the human PXR (HsaPXR) and marine mussel NR1J1 (MgaNR1J1) receptors was observed. Significant differences were observed in all paralogs at 100 nM ATX-A compared to the negative control. Exposure to 100 nM ATX-A resulted in approximately 9and 6-fold increases in the luciferase signals of MgaNR1J1 γ and MgaNR1J1 δ , respectively. In contrast, the induction in the other paralogs studied was lower, reaching up to 4-fold. Notably, no significant differences were observed between the concentrations tested in the human homolog (Figure 1A). Similarly, regarding CYN exposure (Figure 1B), the most sensitive paralogs were MgaNR1J1 γ and MgaNR1J1 δ . At the highest concentration of CYN tested (100 nM), the signal intensity of these paralogs was comparable to that induced by OA. MgaNR1J1 δ exhibited the maximum activity, reaching an approximately 6-fold increase compared to the negative control. In contrast, HsaPXR was the least sensitive paralog. The data obtained from different congeners of Microcystins (MCs) are represented in Figure 2. Overall, MgaNR1J1 paralogs were significantly transactivated in a concentration-dependent manner, while HsaPXR did not respond differently to the concentrations of MCs assayed. Although the results obtained with MC-LR were significantly different, it caused a smaller effect among the congeners studied (Figure 2A). A statistical analysis indicated that the most sensitive paralogs were MgaNR1J1 γ and MgaNR1J1 δ . Moreover, there were no discernible differences between concentrations except for MgaNR1J1 α . Similar trends were obtained when transfected COS-1 cells were exposed to MC-RR (Figure 2B) and MC-YR (Figure 2C). The MgaNR1J1 γ paralog was the most sensitive to the exposure to both toxins at 100 nM, exhibiting an Int. J. Mol. Sci. 2024,25, 6287 8 of 16 increase of up to 9-fold in its activity. Significant differences between the two tested concentrations were observed in all MgaNR1J1 paralogs except for MgaNR1J1 α exposed to MC-YR. 2.2. Marine Toxins The exposure to marine toxins resulted in significant transactivation of HsaPXR and MgaNR1J1 paralogs (Figure 3). Notably, the MgaNR1J1 γ paralog displayed the highest sensitivity to the effects of these toxins, showing an approximately 6-fold induction at the highest tested concentrations, while the MgaNR1J1 β paralog was less responsive. All Nodularin concentrations tested resulted in significant changes compared to the control group (Figure 3A). Moreover, remarkable differences between the concentrations assayed were observed in all groups except for HsaPXR and MgaNR1J1 β . Following exposure to 100 nM Saxitoxin (Figure 3B), similarly to MgaNR1J1 γ , a significantly high induction (approximately 7-fold) comparable to that with the positive control OA was observed in MgaNR1J1 δ , followed by MgaNR1J1 α . HsaPXR and MgaNR1J1 β showed the lowest activities. Among the concentrations tested, no differences were observed between 50 and 100 nM in MgaNR1J1 β and MgaNR1J1 γ . Regarding Tetrodotoxin (Figure 3C), high inductions were obtained with both 50 and 100 nM for the paralog MgaNR1J1 γ , and significant differences between these concentrations were observed only for MgaNR1J1 β and MgaNR1J1δ. 2.3. Natural Compounds The effects on HsaPXR and MgaNR1J1 paralog transactivation by the natural compounds Saint John’s Wort (SJW), Ursolic Acid (UA), and 8-Methoxypsoralene (8M) are shown in Figure 4. The most remarkable effect was the luciferase signal decrease (repression of firefly luciferase expression) with the paralog MgaNR1J1 α exposed to these compounds, indicating that they may act as inverse agonists of this paralog. In the case of SJW (Figure 4A), HsaPXR was the most sensitive (4.43-fold at the highest concentration), with significant concentration-dependent differences compared to the negative control. Significant changes were found for the paralog MgaNR1J1 β at the highest concentration, while the MgaNR1J1 γ paralog was transactivated significantly at 50 and 100 µ g/mL The results obtained with UA are shown in Figure 4B. No significant differences were found among the tested concentrations and the negative control for HsaPXR. Exposure to UA, similarly to that with SJW, led to a significant decrease in luciferase signal for MgaNR1J1 α compared to the negative and positive controls. The same was observed with the lowest test concentration for MgaNR1J1 δ . No significant differences were found in the transactivation of MgaNR1J1 β and MgaNR1J1 γ across all the concentrations assayed. Exposure to 8M (Figure 4C) significantly induced a 3.56-fold increase in luciferase activity with HsaPXR at the highest concentration assayed (100 µ M). Consistent with the other compounds, a significant decrease in luciferase signal was observed across all concentrations tested for MgaNR1J1 α . No significant differences were found in MgaNR1J1 δ . However, the MgaNR1J1 β and MgaNR1J1 γ paralogs had significantly activated luciferase expression with 8M at 100 µM and 50/100 µM, respectively. 2.4. Algal Extracts The transactivation of HsaPXR and MgaNR1J1 α upon exposure to different microalgae extracts is shown in Figure 5. No significant changes in luciferase activity induced by HsaPXR were found after exposure to microalgae extracts compared to the negative control. However, Tetraselmis and Isochrysis extracts significantly increased luciferase activity induced by MgaNR1J1 α , while exposure to LEGE CC 95046 and LEGE CC 91351 extracts had no effects. The highest signal was produced by Tetraselmis extract (Figure 5A) at the highest concentration (100 µ g/mL), with an increase of 3.63-fold induction. Moreover, the transactivation was more evident with the Isochrysis extract (Figure 5B), with significant differences at all concentrations tested. Int. J. Mol. Sci. 2024,25, 6287 9 of 16 3. Discussion Comprehending the xenobiotic metabolism pathways holds significant importance within the field of toxicology due to their influence on the effects of drugs, xenobiotics, and harmful substances. In mammals and other animals, the nuclear receptor PXR plays a crucial role in regulating the expression of drug-metabolizing enzymes, such as cytochrome P450 enzymes, conjugation enzymes, and transporters [ 22 , 23 ]. As a prototypical nuclear receptor, PXR has a DBD at the N-terminus and an LBD at the C-terminus. This LBD shelters a remarkably plastic ligand-binding pocket, giving PXR the ability to recognize a variety of structurally diverse compounds and a single compound in different orientations [ 24 ]. Orthologous genes of PXR (NR1J1) have been reported in marine invertebrates [ 19 , 25 , 26 ], and recently, four orthologous PXR genes were identified in the marine mussel Mytilus galloprovincialis [ 18 , 25 – 27 ]. In the present study, the transfection of four marine mussel NR1J1 paralogs (MgaNR1J1 α , MgaNR1J1 β , MgaNR1J1 γ , MgaNR1J1 δ ) into the COS-1 cell line coupled to a Gal4 luciferase reporter system was used to facilitate the investigation of NR1J1 functionality and the mechanisms behind the activation and regulation of heterologous metabolism and to draw conclusions regarding their distinct responses and sensitivity to the range of studied toxins and their putative roles in bivalves. Bivalve feeding on microalgae often leads, in freshwater environments, to the accumulation of cyanotoxins such as MCs and CYN. Molecular-level changes, including modifications in cytoskeleton proteins, disruptions in energy metabolism, and the induction of xenobiotic metabolism enzymes, have been documented in bivalves exposed to cyanotoxins [ 28 – 30 ]. However, despite the toxic nature of these compounds, bivalves exhibit a remarkable tolerance towards toxin accumulation and their potential adverse effects, likely due to their ability to rapidly metabolize these toxins. In this sense, Oliveira et al. (2020) [ 29 ] concluded that mussels likely possess their own defense mechanisms against cyanotoxins, potentially linked to the overexpression of Enolase 1 (ENO1), Heat Shock Protein 90 (HSP90), and Heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1). Based on the obtained data, the marine mussel NR1J1 receptors are sensitive to both freshwater (ATX-A, CYN, MC-LR, MC-RR, and MC-LR) and marine toxins (NOD, SXT, and TTX), especially to the freshwater cyanotoxins ATX-A, MC-RR, and MC-YR (Figures 1–3). Evidence has been collected indicating that different types of algal toxins are metabolized and undergo chemical conversion in bivalve tissues. For example, MCs bind covalently with glutathione (GSH), reducing the biological activity of the toxin and facilitating its elimination [31–33]. Moreover, this effect was also observed in other aquatic organisms. Li et al. (2013) conducted an experiment in which zebrafish were exposed to crude MCs to examine the role of miRNAs, cyp1A1, and PXR in the toxicity of MCs [ 34 ]. Their findings suggest that MCs alter the transcription levels of the PXR receptor, implying its role in the metabolism and detoxification of these toxins in zebrafish. Furthermore, in line with our own results, the expression of zebrafish PXR receptor was found to be relatively low in the group exposed to lower concentrations (50 µ g/L), while an upregulation was observed in the fish exposed to higher concentrations (200 and 800 µ g/L). Given the high toxicity attributed to this specific congener [ 35 ], our results further imply that one of its mechanisms of action is linked to these paralogs. In the marine environment, bivalves face a different array of compounds, such as OA, saxitoxins, domoic acid, and brevetoxins, primarily produced by dinoflagellates. OA and its analogs (DTXs) undergo esterification with fatty acids of varying structures [ 36 – 39 ] within bivalve tissues, increasing their hydrophilicity and aiding in their elimination [ 36 , 37 ]. Despite the progress in understanding the detoxification of algal toxins, there remains a large gap regarding the molecular processes and enzymes that intervene in and catalyze chemical changes in bivalves. The activation of PXR can differ between species, and PXR may exhibit differential activation and regulation of detoxification pathways in response to distinct classes of toxins [ 26 ]. Accordingly, in the present work, we studied the differential sensitivity of PXR and Int. J. Mol. Sci. 2024,25, 6287 16 of 16 49. Yan, T.; Luo, Y.; Xia, Y.; Hamada, K.; Wang, Q.; Yan, N.; Krausz, K.W.; Ward, J.M.; Hao, H.; Wang, P.; et al. 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