The generation of prooxidant molecules is a consequence of normal cellular functioning (Sies et al., 2017). Eukaryotic cells produce enzymatic and non-enzymatic antioxidant defenses to avoid the oxidative damage that prooxidants could cause in macromolecule (Sies, 1993; Stahl and Sies, 2003). Several external factors can promote an enhanced prooxidant generation (Sies et al., 2017). Previous investigations have shown that metals increase the generation of reactive oxygen and nitrogen species (RS), thus affecting cellular oxidative homeostasis and leading to oxidative imbalance, commonly known as oxidative stress (for a review see Valko et al., 2005; 2007). The oxidative imbalance can damage cells or tissues by lipid peroxidation, protein carboxylation, and even modifications in the DNA (Newman and Unger, 2003).

The apple snail Pomacea canaliculata has emerged as a possible bioindicator of elemental pollution (Adewunmi et al., 1996; Ezemonye et al., 2006; Deng et al., 2008; Vega et al., 2012; Campoy-Diaz et al., 2018; Campoy-Diaz et al., 2020) due to its worldwide distribution in humid tropical and subtropical freshwater ecosystems (Hayes et al., 2015), can be cultured and kept under laboratory-controlled conditions, and their tissues can accumulate a diverse array of ecologically relevant elements (e.g. As, Cd, Cu, Hg, Pb, U, Zn) (Callil and Junk, 2001; Peña and Pocsidio, 2008; Vega et al., 2012; Campoy-Diaz et al., 2018; Huang et al., 2018; Campoy-Diaz et al., 2020; Juarez et al., 2022). Furthermore, the digestive gland and symbionts from P. canaliculata participate in metal accumulation. Additionally, these symbionts are involved in metal depuration (Castro-Vazquez et al., 2002; Vega et al., 2005; Vega et al., 2006; Vega et al., 2012; Campoy-Diaz et al., 2018; Campoy-Diaz et al., 2020) when are expelled in the feces (Koch et al., 2006). However, our knowledge about the physiological response of the digestive gland to different prooxidant elements remains incomplete.

The high persistence, wide environmental occurrence, and proven toxicity of metals make them an urgent issue requiring priority study. Nieboer and Richardson (1980) have emphasized the biochemical basis for metal-ion toxicity, together with the ecotoxicological and ecophysiological significance for organisms and ecosystems exposed to these elements. They classify metals by their differential affinities for functional biological groups: A class elements seek oxygen (O), B class elements seek nitrogen (N) and sulfur (S), and intermediate class elements seek either O, N, or S. In this study, we have selected three elements included in the first 100 substances of the priority list of Agency for Toxic Substances and Disease Registry: U from the A-class, Hg from the B-class, and As from the intermediate-class. By including these elements with different biochemical affinities for biomolecules, we evaluated the balance between free radicals’ production and antioxidant response, as well as oxidative damage in the digestive gland of P. canaliculata after elemental exposure.

Elemental concentrations of Hg, As, and U in digestive gland were significantly higher in exposed snails than in non-exposed snails (Student’s t-test, p < 0.05; Figure1). Bioconcentration factors of Hg, As, and U were 25, 23, and 53, respectively.

To characterize the oxidative state, we first evaluated the production of RS (Figure 2). This parameter significantly increased in the first 24 h after elemental exposure (Figure 2A–C, respectively), compared to group control (0 h), and it remained significantly high until the end of the experimental period (72 h for Hg and 96 h for As and U).

Besides, the characterization of the oxidative state was completed with the evaluation of non-enzymatic and enzymatic antioxidants defenses. Figure 3 resumes the involvement of non-enzymatic antioxidant defenses after elemental exposure. In snails exposed to Hg, the α-T and β-C levels did not change significantly (GLM, p > 0.05; Figure 3A, D). In As-exposed animals, α-T level remained unchanged (GLM, p > 0.05; Figure 3B) but β-C concentration fell significantly in the first 24 h, compared to control (0 h), then recovered to levels close to basal at 48 h to end up falling until the end of the exposure again (Figure 3E). In U-exposed snails, α-T and β-C concentrations were consumed early (24 h) and remained significantly low until 96 h (Figures 3C, F). Digestive gland showed a significant increase in MTs after elemental exposure (Figures 3G–I): 24 and 72 h in Hg-exposed snails, 24 h in As-exposed snails, and 24, 48 and 72 h in U-exposed snails. Total uric acid concentrations (Figures 3J–L) decreased significantly after 24 h exposure to Hg, As, and U, and remained low until the end of the exposure time (96 h). Figure 4 shows the enzymatic antioxidant defenses after elemental exposure. Both enzymatic activities, CAT and GST, remained unchanged in Hg-exposed (GLM, p > 0.05; Figures 4A, D) and As-exposed (GLM, p > 0.05; Figure 4B, E) snails. In U-exposed animals, CAT activity increased significantly at 72 h, compared to other endpoints, (Figure 4C), while GST activity dropped abruptly at 24 h (Figure 4E) and it began to increase continuously until the end of the exposure (72 and 96 h); although this increase fails to restore its pre-exposure values.

Additionally, we evaluated if the antioxidant protection mechanisms were sufficient to counteract RS production after elemental exposure through the occurrence of damage to biomolecules such as lipids and proteins (Figure 5). The generation rate of LR• and carbonyl groups were higher in exposed animals compared to control group (0 h), but statistical significance was reached only at some endpoints. The GLMs analysis showed significantly higher values of LR• for Hg, (96 h > 0, 24, 48, 72 h), As (96 h > 24 and 48 h > 0 h), and U (24 h > 48 and 72 h > 0 h), and of carbonyl groups for Hg (24–96 h > 0 h), As (24 and 96 h > 24 and 48 h > 0 h), and U (24 h > 48 and 72 h > 0 h).

The central objective of the current work was to assess changes in the oxidative status and lipids and proteins oxidation in the digestive gland of the gastropod Pomacea canaliculata after an aquatic exposition for 96 h to ecologically relevant concentrations of Hg, As, and U.

The animals did not show mortality during exposure, despite the large concentrations of these elements, which are toxic to aquatic ecosystems and human health. Our preceding studies (Vega et al., 2012; Campoy-Diaz et al., 2018; Juarez et al., 2022) showed that the digestive gland accumulates metals of class A, B, and intermediate from water. Moreover, digestive epithelial cells may accumulate elements directly by endocytosis after food digestion in the stomach (Juarez et al., 2022). Herein, the digestive gland bioconcentrates the three non-essential elements, generating early reactive species thus causing protein and lipid oxidation. Time changes in the lipid peroxidation and protein damage differed among the three elements used (Figure 5). It is likely that this cellular response is associated with the affinities of Hg, As, and U for different functional biological groups or with different degrees of effectiveness of antioxidant defenses. Inorganic Hg and As have a high affinity for reduced sulfur groups (Sharma et al., 1993; Quig, 1998; Ravichandran, 2004; Shen et al., 2005; Maltez et al., 2009), depleting intracellular reserve of thiols (Valko et al., 2005). Furthermore, As and U (in the form of uranyl ion) are prooxidant elements that may respectively increase the superoxide anion and hydrogen peroxide levels (Shi et al., 2004), hydroxyl radicals, and hydrogen peroxide (Garmash et al., 2014), thus producing oxidative stress and macromolecules damage (Barescut et al., 2005; Bergmann et al., 2018). Additionally, uranyl may bind to nucleotides through their phosphoric groups, competing with calcium and magnesium ions, which cause a reduction of bioavailable ATP for different metabolic processes (De Stefano et al., 2005). Besides, U may deplete reduced glutathione, as was reported in isolated hepatocytes from laboratory rat (Rattus norvegicus, Pourahmad et al., 2011) and zebrafish (Danio rerio, Barillet et al., 2011).

Given that the three elements produce an oxidative imbalance in the digestive gland of P. canaliculata, snails may effectively delay or attenuate lipid peroxidation and protein damage activating its cellular antioxidant machinery, as occurs against other environmental stressors such as low temperature, desiccation, or xenobiotics (Giraud-Billoud et al., 2011; Giraud-Billoud et al., 2013; Giraud-Billoud et al., 2018; Giraud-Billoud et al., 2022). In the next paragraphs, we discuss the importance and effectiveness of each antioxidant defense during oxidative imbalance.

α-T and β-C are lipophilic dietary molecules currently known in mollusks, with behavioral, physiological, and protective functions (Tsushima et al., 1997; Dreon et al., 2004; Rivera-Ingraham et al., 2013; Abdullah and Seulalae, 2020). These molecules are often associated with cell membranes and prevent lipid peroxidation by neutralizing specific reactive species (Neely et al., 1988; Sies and Stahl, 1995) or even interrupting the sequential oxidation reactions of polyunsaturated fatty acids (Scarpa et al., 1984; Sies and Stahl, 1995). Herein, the consumption of α-T and β-C was variable among snails exposed to three elements. U-exposed snails showed an early and high consumption of both molecules, counteracting partially the oxidative burst and delaying the lipid peroxidation. In As-exposed snails, the consumption of β-C was around 67%, but that of α-T was not significant during the entire experimental period, which could be indicating a compartmentalization of the oxidative process. Interestingly, Hg-exposed snails maintained the levels of α-T and β-C without changes in lipid peroxidation during the first 72 h. These findings may be associated with the fact that the digestive gland of P. canaliculata is the main organ involved in the acquisition, accumulation, and metabolization of different dietary carotenoids (Tsushima et al., 1997), and that the individuals were fed throughout the experimental period (this work).

MTs are cytosolic non-enzymatic cysteine-rich proteins virtually present in all animals (Blindauer and Leszczyszyn, 2010) that regulate the availability of essential metals (Zn and Cu), and detoxify non-essential elements (Amiard et al., 2006). In this work, high levels of MTs were reached after elemental exposure, but with different kinetics. It is likely that these cellular changes are associated with a differential expression, as was reported in the apple snails Pomacea bridgesii and Marisa cornuarietis exposed to intermediate and B-class elements (Maltez et al., 2009). Also, the MTs variations may be a consequence of their lysosomal compartmentalization (Hamer, 1986) for its subsequent degradation and excretion (Marigomez et al., 2002). This evaluation should be considered a preliminary appraisal and a complete one should evaluate the transcription of two MTs codifying genes found in the genome in P. canaliculata (Liu et al., 2018; Sun et al., 2019).

P. canaliculata accumulates urate crystalloids in the perivascular tissue of the digestive gland (Vega et al., 2007; Giraud-Billoud et al., 2008). Previous evidence has shown that uric acid-producing cells exhibit an active turnover between asynchronous formation and lysis of crystalloids (Giraud-Billoud et al., 2008). Furthermore, uric acid is an antioxidant in this species, used after long periods of dormancy induced by estivation or hibernation to tolerate the oxidative damage induced by arousal (Giraud-Billoud et al., 2013; Giraud-Billoud et al., 2018). In this work, total uric acid decreased approximately 3-fold 24 h after exposure to Hg, As, and U, and it did not recover the initial levels, which may indicate an increase in crystalloid lysis and its use as a non-enzymatic antioxidant to face the oxidative burst. It is possible that uric acid and carotenoids act as the first line of defense against the increase in RS induced by exposure to metals, to prevent lipid peroxidation of cell membranes.

CAT is an antioxidant enzyme that catalyzes the reduction of intracellular H₂O₂ to water and oxygen (Orbea et al., 2000), thus counteracting the activity of H₂O₂-generating oxidases. The role of CAT, as an enzymatic antioxidant protective mechanism in P. canaliculata, has recently been characterized in models of acute stress where reactivation after hypometabolism induced by estivation or hibernation is related to increases in its expression and activity (Sun et al., 2019; Giraud Billoud et al., 2022; Rodriguez et al., 2023). However, the enzymatic activity of CAT remains approximately constant (with some small variations in U-exposed snails), even though the non-enzymatic antioxidant defenses partially counteract RS and protein damage. It is still possible that the protein synthesis or CAT activity may be affected by the presence of metals in the digestive gland cells. The GST family includes cytosolic enzymes that conjugate electrophile groups with reduced glutathione for detoxification (Bhagat et al., 2016). These enzymes do not seem to have a functional role in snails exposed to the three elements studied. Moreover, the GST activity falls in U-exposed snail, which may be associated with early protein damage. Together, these findings could be indicating that CAT and GST are not necessary at the early stages of the oxidative process induced by metals.

The freshwater gastropod P. canaliculata bioconcentrated Hg, As, and U at high levels in the digestive gland when exposed for 96 h at ecologically relevant concentrations. These elements generated early RS, lipid peroxidation, and the protein damage and redox disbalance were partially compensated by non-enzymatic antioxidant defenses. The consumptions of α-T, β-C, and uric acid were variable among snails exposed to three elements. MTs increased at similar levels after exposure but with different kinetics. CAT and GST were not necessary for the early stages of the oxidative process by metals. The bioconcentration factors and biochemical parameters studied in this work confirm that this species may be used as a bioindicator of metal pollution in freshwater bodies. Two ecotoxicological considerations related to human and animal health should be noted: 1) the direct consumption of apple snails by humans or their use as feed for pigs, and 2) the possible biomagnification of metals in ecosystems, where birds almost exclusively consume these snails.