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Effects of chlorothalonil on the antioxidant defense system of mussels Perna perna
Ecotoxicology and Environmental Safety 190 (2020) 110119
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Effects of chlorothalonil on the antioxidant defense system of mussels Perna perna
T
Amanda da Silveira Guerreiroa,∗, Fiamma Eugênia Lemos Abreub, Gilberto Fillmannb, Juliana Zomer Sandrinia a
Programa de Pós-Graduação em Ciências Fisiológicas, Instituto de Ciências Biológicas, ICB, Universidade Federal do Rio Grande - FURG, 96203-900, Rio Grande, RS, Brazil b Programa de Pós-Graduação em Oceanologia, Instituto de Oceanografia, IO, Universidade Federal do Rio Grande - FURG, 96203-900, Rio Grande, RS, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Antifouling biocides Antioxidant enzymes Bivalve Biomarker Oxidative stress Toxicity
Chlorothalonil is an effective fungicide used in agriculture and formulations of antifouling paints, which use and possible toxicity has been generating great concern. Thus, the present study investigated the effects of chlorothalonil on the antioxidant defense system (ADS) of the mussel Perna perna. The ADS was evaluated in gills and digestive gland after 24 h and 96 h of exposure to environmental relevant levels of chlorothalonil (0.1 and 10 μg/ L). The activity of the enzymes superoxide dismutase (SOD), catalase (CAT), glutamate cysteine-ligase (GCL) and glutathione S-transferase (GST), levels of non-enzymatic defenses, represented by glutathione (GSH), and lipoperoxidation (LPO) and protein carbonyls (PCO) were evaluated. Results indicated that exposure to chlorothalonil is affecting the ADS in both tissues. While the activity of SOD increased and GST and GSH were not altered in gills, they decreased in digestive gland after 24 h of exposure to 10 μg/L of chlorothalonil. The contrasting results indicate that gills and digestive gland presented different patterns of responses after exposure to chlorothalonil. Moreover, a tissue-specific response to chlorothalonil was observed. Gills could be acting as the first line of defense, presenting higher enzymatic levels with minor effects on the parameters analyzed. On the other hand, digestive gland, with lower levels of antioxidant defenses, was the most affect organ by chlorothalonil. It also should be highlighted that the fungicide reduced the glutathione metabolism in the digestive gland, which can lead to an imbalance of the redox state within the cells of animals.
1. Introduction
commonly used since they are considered less toxic and less persistent in the environment (Konstantinou and Albanis, 2004; Thomas and Brooks, 2010; Voulvoulis et al., 2002). Chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile) is an aromatic halogen compound that was first introduced as a broad-spectrum fungicide in agriculture (Ernst et al., 1991). Due to its antifungal properties, it was also introduced as a booster biocide in commercial antifouling paints as well. Environmental levels ranging from 0.004 to 1.38 μg/L were already reported for marine and freshwater ecosystems, demonstrating its dual usage (Edwards et al., 2017; Lee et al., 2010; Voulvoulis et al., 2000). In the Korean coast, Lee et al. (2010, 2015) reported concentrations close to 0.067 μg/L in water and 50.6 ng/L in sediment samples and associated those values with the increased activity of the harbors nearby. Levels of the contaminant were also found in rivers, which is correlated with the agricultural activity of close areas (Callicott and Hooper-Bùi, 2019). Those levels were ranging from 0.0035 μg/L to 0.024 μg/L and were observed in superficial waters of
Marine biofouling is a natural phenomenon that often poses a problem for the shipping industry. Biofouling, defined as the accumulation of microorganisms, plants, and aquatic invertebrates on surfaces immersed in seawater, can increase the roughness and frictional resistance of ships, leading to an increase in fuel consumption and docking frequency (Omae, 2003; Yebra et al., 2004). To prevent the settlement and growth of organisms on those surfaces, antifouling paints have been developed and used extensively. Organotin-based compounds, such as tributyltin (TBT), were the most successful against fouling (Omae, 2003). However, these compounds were banned due to their persistence and toxicity (Bao et al., 2011; Bigatti et al., 2009; Evans et al., 2000; Ofoegbu et al., 2016; Strand et al., 2009). In this context, safer alternatives to TBT have been developed and introduced in paint formulations. Booster biocides, such as copper-pyrithione, zinc-pyrithione, DCOIT, Irgarol 1051, diuron, and chlorothalonil, are some of the most
∗
Corresponding author. E-mail address: [email protected] (A.d.S. Guerreiro).
https://doi.org/10.1016/j.ecoenv.2019.110119 Received 11 September 2019; Received in revised form 16 December 2019; Accepted 20 December 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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every two days and then, water was renewed.
the Mississippi river (Callicott and Hooper-Bùi, 2019). Information concerning chlorothalonil contamination levels in the coast of Brazil and other Latin American countries is still not available. Some studies have demonstrated that chlorothalonil can be harmful to the aquatic biota, inducing DNA damage (Cima et al., 2008), and causing growth abnormalities (Bellas, 2006) and mortality in different marine species (Ernst et al., 1991; Key et al., 2003; Koutsaftis and Aoyama, 2007). For bivalves, a 96 h EC50 of 8.8 μg/L (Bellas, 2006) and a 96 h LC50 of 5900 μg/L (Ernst et al., 1991) was reported for larvae and adults of Mytilus edulis, respectively, exposed to chlorothalonil or exposed to a formulated product (BRAVO 500) containing chlorothalonil. In the ascidian Bothryllus schollosseri, exposure to chlorothalonil resulted in loss of function of mitochondria, lower ATP production, and subsequent apoptosis (Cima et al., 2008). In hemocytes of the mussel Perna perna, exposure to chlorothalonil caused decrease in cellular viability and increase in cellular adhesion and phagocytic activity (Guerreiro et al., 2017). It has been proposed that this biocide has the ability to bind to sulfhydryl groups of peptides and proteins, which could lead to effects like reduction of glutathione levels and inhibition of enzymes (i.e. NADPH oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) (Baier-Anderson and Anderson, 2000; Tillman et al., 1973; Long and Siegel, 1975). Indeed, some authors (Davies, 1985; Gallagher et al., 1991) have demonstrated that the tripeptide glutathione (GSH) and the enzyme glutathione S-transferase (GST) are important in chlorothalonil metabolism and toxicity. They observed the formation of chlorothalonil-glutathione conjugates in fish species and suggested that GST has an important role in chlorothalonil toxicity and detoxification. Considering that GSH and GST are important in the biotransformation process, as well as in the antioxidant defense system, studies have proposed that, similar to many other environmental pollutants, chlorothalonil could induce oxidative stress by altering the balance between those molecules (Cima et al., 2008; Tillman et al., 1973; Baier-Anderson and Anderson, 2000). Recently, Barreto et al., (2018) observed that chlorothalonil induced oxidative stress in the estuarine polychaeta Laeonereis acuta through reduction of its total antioxidant capacity and alteration in glutathione metabolism, observed as altered activities of glutamate cysteine-ligase and glutathione S-transferase. In this context, chlorothalonil might impair the antioxidant defense systems of other organisms by changing the redox status of cells. Seeing the hazard of chlorothalonil exposure to aquatic organisms, the present study aimed to investigate its effects on the antioxidant defense system of the mussel P. perna. Mussels have been widely used in biomonitoring programs (Goldberg, 1986; Goldberg and Bertine, 2000) and species, such as P. perna, are widely distributed along the Brazilian coast (Siddall, 1980). In addition, mussels allow an assessment of environmental contaminants partitioned either in the dissolved (by respiration) and particulate (by filter-feeding) phases of water column (Dame, 1996). In this context, the antioxidant defense system of P. perna was investigated after exposure to the biocide chlorothalonil. The concentrations tested in the present study (0.1 μg/L and 10 μg/L) are environmentally relevant and considered sublethal to P. perna.
2.2. Experimental design Mussels (N = 72) were separated into nine 6-L tanks to perform the experiment in triplicates (three aquaria for each treatment). The water conditions described previously were maintained until the end of the experimental period. Animals were exposed to the biocide chlorothalonil for up to 96 h at the following nominal concentrations: control (with 0.01% dimethyl sulfoxide), and 0.1 μg/L (0.37 nM) and 10 μg/L (37 nM) of chlorothalonil. After 24 h and 96 h of exposure, 6 mussels of each treatment (2 of each aquaria) were dissected, and gills and digestive gland were collected. Tissues were stored at −80 °C until further analyses. Concentrations of chlorothalonil in water were measured as described by Barreto et al. (2018). Briefly, water samples were extracted by SPE (C18 cartridge) and the eluates analyzed by gas chromatography/electron capture detector (GC-ECD). Measurements were conducted immediately after contaminant addition (0 h) and after 48 h of exposure. 2.3. Biochemical biomarkers For enzymatic measures of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) activity, mussel tissues (gills and digestive gland) were homogenized (1:4 w/v) in cold buffer (20 mM Tris base, 1 mM EDTA, 0.5 M sucrose, 0.15 M KCl, and 0.1 mM PMSF, pH 7.6). Samples were centrifuged at 1000 g for 20 min at 4 °C and subsequently at 10,000 g for 45 min at 4 °C. Supernatant was collected while the resulting pellet was discarded. Protein concentration was evaluated by the biuret method. SOD activity was measured in a spectrophotometer at 550 nm according to McCord and Fridovich (1969). In this assay, the reduction of cytochrome C was evaluated through the inhibition of xanthine:xanthine oxidase. One SOD unit represents the amount of enzyme necessary for 50% inhibition of cytochrome C at 25 °C. CAT activity was evaluated through the decomposition of H2O2 per minute at 240 nm, as described by Beutler (1975), and was expressed as CAT units. One CAT unit represents the amount of enzyme able to decompose 1 μmol of H2O2 per minute per milligram of protein. GST activity was measured at 340 nm, using 1-chloro-2,4-dinitro- benzene (CDNB, Sigma) as a substrate for its activity (Habig et al., 1974). The results were expressed as GST units, where one GST unit represents the amount of enzyme necessary to conjugate 1 μmol of CDNB per minute per milligram of protein at 25 °C. Considering glutathione metabolism, glutamate cysteine-ligase (GCL) activity and glutathione (GSH) content were measured following the protocol of White et al. (2003). Mussel tissues (gills and digestive gland) were first homogenized (1:5 w/v) in cold buffer (100 mM TrisHCl, 2 mM EDTA, 5 mM MgCl2.6H2O) and centrifuged at 20,000 g for 20 min at 4 °C. The pellet was then discarded, and the supernatant was stored at −80 °C. Extracts were then evaluated following the reaction of the fluorescent compound 2,3-naphtalenedicarboxaldehyde (NDA) with GSH and with γ-glutamyl-cysteine residues. The reaction product was analyzed in a fluorimeter (Victor, PerkinElmer) at 472 nm (excitation) and 528 nm (emission). The GCL activity was expressed as ηmol of GSH per hour and per mg of protein, while the GSH concentration was expressed as mg of GSH per mg of protein.
2. Materials and methods 2.1. Animals P. perna were obtained from a mariculture farm located in the southwest of Santa Catarina Island, Brazil (−27.729,769, −48.562,973). Animals were transported to the Universidade Federal do Rio Grande – FURG, where the experiments were conducted. Thereafter, mussels were acclimated for 15 days in tanks (20 L) with filtered (5 μm) seawater (salinity of 30), under constant aeration, temperature of 20 ± 2 °C, and photoperiod of 12L:12D. Animals were fed with phytoplankton Nannochloropsis sp and Conticribra weisfloggii in
2.4. Quantification of lipoperoxidation levels (LPO) Lipid peroxidation was evaluated through the TBARS assay, according to Oakes and Van der Kraak, (2003). The end products of lipid peroxidation were measured considering the reaction of substances, mainly malondialdehyde, with thiobarbituric acid. In this assay, samples were homogenized (1:5 w/v) in cold buffer (100 mM Tris-HCl, 2
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0.1 μg/L and 10 μg/L of chlorothalonil for 24 h (Fig. 3A), but no effect was observed after 96 h of exposure. No alterations were observed in the digestive gland (Fig. 3B). Although no effects were observed in gills (Fig. 3C), a decrease in levels of protein carbonyls was detected in digestive glands exposed to 10 μg/L of chlorothalonil for 24 h and 96 h (Fig. 3D).
2 mM EDTA, 5 mM MgCl2.6H2O) and centrifuged at 20,000 g for 20 min at 4 °C. The supernatant was incubated with thiobarbituric acid, butylated hydroxytoluene, acetic acid and 8.1% SDS buffer and, then, heated to 95 °C for 30 min to promote a color reaction. Analyses were carried out in a fluorimeter (Victor, PerkinElmer) at 515 nm (excitation) and 553 nm (emission). The results were expressed as nmol MDA per mg protein.
4. Discussion 2.5. Protein carbonyl content (PCO) Despite the economic importance to naval industry, it is known that biocides presented in antifouling paints may also cause toxicity to nontarget species. Diuron and Irgarol, for example, are considered extremely harmful to marine phytoplankton (Bao et al., 2011; DeLorenzo and Fulton, 2012). Both biocides are known photosynthetic system II inhibitors, and can affect cyanobacteria, algae and the symbiotic dinoflagellates presented in corals (Jones and Kerswell, 2003). DCOIT also has effects on algae development and growth (Onduka et al., 2013), and studies clearly demonstrates its toxic potential to fish and polychaetas as well (Chen et al., 2016; Onduka et al., 2013). Chlorothalonil, on the other hand, is a fungicide widely applied in agricultural sites and as a co-biocide in antifouling paints that still lacks information concerning its toxic effects on terrestrial and aquatic species. A previous study conducted by our group had already demonstrated sublethal effects in the immune system of mussel P. perna after exposure to 0.1 μg/L and 10 μg/L of chlorothalonil (Guerreiro et al., 2017). Those concentrations might be representative of the actual scenario in many aquatic ecosystems, since similar values have already been reported worldwide. Lee et al. (2010) and Sakkas et al., 2002b, for example, have found chlorothalonil levels as high as 0.07 μg/L and 0.06 μg/L in water of harbor areas from Korea and Greece, respectively. Other studies reported even higher concentrations (1.38 μg/L) in estuarine areas of the UK, which correlates with the end of local boating season (Voulvoulis et al., 2000). Chlorothalonil was also reported in sediments at concentrations up to 690 ng/g, impacting both nektonic and benthic organisms (Albanis et al., 2002; Lee et al., 2015). Although many studies have been detecting chlorothalonil in water and sediments, this compound appears to present a relatively rapid degradation and/or metabolization in the environment (Davies, 1985; Gallagher et al., 1991; Sakkas et al., 2002a). Since chlorothalonil is expected to degrade within 48 h in natural seawater (Sakkas et al., 2002a), water treatments were renewed every two days during the present study. Even so, measured chlorothalonil levels in water were < LOD at all treatments after 48 h which might be due to the uptake and/or metabolization by mussels. As filter-feeding organisms, mussels not only remove materials from the water column, but they can induce nutrient cycling in the environment (Dame, 1996). Also, they are capable of accumulating many types of contaminants in their soft tissues (Sehonova et al., 2018; Viarengo and Canesi, 1991), such as metals (Estrada et al., 2017), hydrocarbons (Lüchmann et al., 2011), pesticides (Pariseau et al., 2009), and other organic compounds (Varol and Sünbül, 2017; Quintas et al., 2017), which can induce several kinds of damage related to contaminant exposure and accumulation. Results from Davies and White (1985) suggest that chlorothalonil can accumulate in fish tissues. However, for bivalve species, such as the mussel P. perna, information regarding chlorothalonil accumulation and metabolization still need a thorough assessment. In any case, accumulating or not, exposure to chlorothalonil causes several types of damage to organisms (Barreto et al., 2018; Cima et al., 2008), including to the immune system of mussels (Guerreiro et al., 2017). Some studies have demonstrated that biotransformation of chlorothalonil in marine and terrestrial organisms is mainly through the conjugation with glutathione (Kim et al., 2004; Rosner et al., 1996). In rats, Rosner et al., (1996) observed that chlorothalonil was rapidly transformed to 4,6-bis(glutathion-S-yl)-2,5-dichloroisophthalonitril in
Protein carbonyl content was determined using the OxySelect™ Protein Carbonyl Fluorimetric Assay Kit (MyBioSource). Briefly, samples were homogenized (1:4 w/v) in cold buffer (20 mM Tris base, 1 mM EDTA, 0.5 M sucrose, 0.15 M KCl, and 0.1 mM PMSF, pH 7.6) and centrifuged twice (1000 g for 20 min at 4 °C and subsequently at 10,000 g for 45 min at 4 °C). Supernatants were then incubated with the OxiSelect™ fluorophore. After the incubation period, 20% trichloroacetic acid solution was added to induce the precipitation of proteins. Free fluorophore was then removed by acetone washes. Absorbance was measured fluorometrically with a 485/538 nm filter. The results were expressed as nmol per mg of protein. 2.6. Statistical analysis Results are presented as mean ± standard error. Data were tested for normality and homoscedasticity and, when complied with the assumption, one-way ANOVA followed by Tukey HSD post hoc test were applied. For nonparametric data, Kruskal-Wallis test followed by the post-hoc of multiple comparisons of the mean ranks were applied for all groups. Statistical analyses were performed using Statistic 7 (StatSoft) with a significance level of 5% (p < 0.05). 3. Results Despite no mortality was observed, exposure to both concentrations of chlorothalonil (0.1 μg/L and 10 μg/L) altered the parameters assessed of the antioxidant defense system of mussels. Chlorothalonil concentrations evaluated in each treatment were 0.1 μg/L (nominal concentration of 0.1 μg/L) and 6.5 μg/L (nominal concentration of 10 μg/L) at 0 h. For the control treatment, the measured chlorothalonil concentration was lower than the limit of detection (LD < 0.1 μg/L). Water samples collected at 48 h were also analyzed to quantify chlorothalonil at the end of treatments. For each treatment, including the control group, the measured chlorothalonil concentration was lower than the limit of detection (LD < 0.1 μg/L). Concerning SOD activity, an increase was observed in gills exposed to the highest concentration of chlorothalonil (10 μg/L) for 24 h. This effect was not observed after 96 h of exposure (Fig. 1A). Despite this increase in gills, a decrease in its activity was observed in digestive gland exposed to both concentrations tested for 24 h. Again, this response was not observed at the end of the experimental period (96 h) (Fig. 1B). The activity of catalase was not significantly altered by any of the concentrations tested in gills (Fig. 1C) or digestive gland (Fig. 1D) during either of the experimental periods. Regarding GST activity, no effects of chlorothalonil were observed in gills after 24 h or 96 h of exposure (Fig. 1E). However, a decrease in the activity of this enzyme was observed in digestive gland exposed to both treatments for 24 h. This effect was not observed after 96 h of exposure (Fig. 1F). A decrease in GCL activity was observed in gills of animals exposed to 10 μg/L for 24 h and to 0.1 μg/L and 10 μg/L of chlorothalonil for 96 h (Fig. 2A). In the digestive gland, an increase in GCL activity was observed in animals exposed to 0.1 μg/L of chlorothalonil for 96 h (Fig. 2B). On contrary, GSH levels were not significantly altered in the gills (Fig. 2C), while a reduction in levels of glutathione was observed in digestive gland exposure to 10 μg/L of the biocide for 24 h (Fig. 2D). Levels of lipid peroxidation (as TBARS) reduced in gills exposed to 3
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Fig. 1. Activity (mean ± standard error, N = 72) of superoxide dismutase (SOD) (A and B), catalase (CAT) (C and D), and glutathione S-transferase (GST) (E and F) in gills (left) and digestive gland (right) of mussels P. perna exposed to 0.1 μg/L and 10 μg/L of chlorothalonil for 24 h and 96 h. Different letters indicate statistically significant differences (p < 0.05).
of NADPH oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has been discussed (Long and Siegel, 1975; Baier-Anderson and Anderson, 2000). Glutathione (GSH), a known target of chlorothalonil, is a tripeptide that contains a sulfhydryl group due to the presence of a cysteine residue and participates in the antioxidant defense system of organisms in order to maintain the redox state of cells (Regoli and Giuliani, 2014). Moreover, it is an important agent in cellular detoxification processes, since it can be conjugated with endogenous or exogenous compounds (Meister and Anderson, 1983). In this context, GSH can intercept electrophilic and oxidant species, preventing damage to nucleic acids and proteins (Pompella et al., 2003; Trevisan et al., 2016). Elevated GSH levels are usually related to an
the presence of glutathione in the rat liver cytosol. Mono-glutathione conjugates are observed as intermediates in the enzymatic reaction, while tri-glutathione conjugates are the final metabolite in the biotransformation of chlorothalonil (Kim et al., 2004). This might indicate that the toxicity of the compound is related with its own biotransformation process and that might be related with the altered antioxidant pathways. The underlying mechanisms involved in chlorothalonil toxicity are still under investigation. Nevertheless, studies have already demonstrated that this compound has binding affinity for the sulfhydryl groups of peptides and proteins (Tillman et al., 1973). Its involvement in cellular respiration and in the glycolytic pathway, through inhibition 4
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Fig. 2. Glutamate cysteine-ligase (GCL) activity (A and B) and glutathione (GSH) (C and D) content (mean ± standard error, N = 72) in gills (left) and digestive gland (right) of mussels P. perna exposed to 0.1 μg/L and 10 μg/L of chlorothalonil for 24 h and 96 h. Different letters indicate statistically significant differences (p < 0.05).
would be important for the protection of organisms against chlorothalonil toxicity, since this compound is mainly metabolized through glutathione conjugates (Gallagher et al., 1991; Rosner et al., 1996). The present results, however, suggest that decreases in GST activity might be correlated with an increase in biotransformation processes and/or the inability of the animal in producing enough GST to cope with the contaminant exposure, at least in the period evaluated. As observed by Gallagher et al., (1991) and by Rosner et al., (1996), GST involvement in chlorothalonil biotransformation is clear, therefore, its decreased activity may be causing toxicity to the animals. Both SOD and CAT also participate in the antioxidant defense system of organisms, protecting cells from oxidative damage caused by the exposure to contaminants (Livingstone, 2001). According to Escobar et al., (1996), alterations in SOD activity might even be related to production of reactive oxygen species (ROS). Some authors suggest that elevated ROS levels may inhibit some antioxidant enzymes, such as SOD (Escobar et al., 1996), while others suggest that the increase in ROS production might be the signal to drive antioxidant responses (Zhao et al., 2017). Although not evaluated in the present study, ROS levels might impact the enzymes involved in the antioxidant defense system, as previously commented. SOD activity was increased in the gills and decreased in the digestive gland after 96 h of exposure to chlorothalonil, and such a difference may be related to organ specificity. Considering that gills are the major site of uptake of chemicals from water (Hayton and Barron, 1990), the increase observed in SOD activity
increase in capacity to resist oxidative stress, while reduced levels might lead to an increase in oxidative damage (Faggio et al., 2016; Gobi et al., 2018). As observed by Davies, (1985) and Gallagher et al., (1991), chlorothalonil targets GSH and promotes a marked decrease of the tripeptide level. However, the decrease in GSH in fish is followed by an increase in GST activity, leading to the formation of mono-, di-, and tri-glutathione/chlorothalonil conjugates. In the present study, alterations in GSH levels were observed in digestive gland only. Although no effects were observed in gills, levels of GSH were at least 3 times higher than in digestive gland. These decreases observed in digestive gland might be related to chlorothalonil's biotransformation within the tissues. Considering that gills are key players in bivalve defenses, they can act as a metabolic barrier against an electrophilic burden, thereby proving a stronger defense against many contaminants (Ahmad et al., 2011; Trevisan et al., 2014, 2016). In addition to GSH, chlorothalonil was responsible for altering levels of other enzymes related to glutathione metabolism. The activity of GCL, the main enzyme responsible for GSH biosynthesis through the binding of glutamic acid and cysteine (Regoli and Giuliani, 2014), was decreased in the gills after 24 h and 96 h of exposure. Despite that, this decrease does not seem to interfere with the GSH levels observed. An increase in GCL activity, however, was observed only in polychaetas exposed to 100 μg/L of chlorothalonil for 24 h (Barreto et al., 2018), which was 10 times higher than those used in the present study. It is known that under different environmental stress situations, GSTs can be altered (van der Oost et al., 2003). Increases in GST activity 5
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Fig. 3. Lipoperoxidation (as TBARS) (A and B) and protein carbonyl (C and D) levels (mean ± standard error, N = 72) in gills (left) and digestive gland (right) of mussels P. perna exposed to 0.1 μg/L and 10 μg/L of chlorothalonil for 24 h and 96 h. Different letters indicate statistically significant differences (p < 0.05).
in gills and may be correlated with higher basal levels of most of the enzymes analyzed. As reported by Baier-Anderson and Anderson (2000), a suppression in superoxide production, evaluated indirectly by the increase in the activity of SOD, may be an explanation for the decrease in the damage of gills. For the PCO results, a decrease in digestive gland of the organisms exposed to chlorothalonil was observed. Since chlorothalonil targets the antioxidant defenses of organisms, it can be suggested that the main antioxidant pathway, driven by the Nrf 2 (nuclear factor erythroid 2 – related factor 2), is modulating the activation of protective agents, such as α-tocopherol. Indeed, Guerreiro and colleagues (manuscript submitted to publication) showed that chlorothalonil affects most of AhR and Nrf2-targeted genes, such as GSTs, SOD and CYP1A2, including AhR own expression. Therefore, the toxicity of chlorothalonil can be correlated with alterations in the antioxidant pathways, which are extremely important for avoiding oxidative stress situations. It is important to highlight that basal levels of enzymatic activity, as well as levels of PCO, were quite different among the tissues analyzed. In general, digestive gland was the most impaired tissue. Basal levels regarding GSH and PCO content demonstrated that higher oxidation in proteins and lesser defenses are frequently observed in digestive gland. Gills, on the other hand, present higher levels of GSH and other antioxidant enzymes. These results corroborate with the role of gills as a metabolic barrier against xenobiotic exposure, and of digestive gland as a more vulnerable tissue in front of exposure to toxicants. This vulnerability makes this tissue important in ecotoxicology analyses, as evaluated by Faggio et al., (2018).
at this tissue may be important since indicate a better protection against ROS formation. Haque et al., (2019) also demonstrates that an increase in the activity of this enzyme is expected for other bivalve species, such as Crassostrea gigas and Mytilus edulis. Those authors reported that increases in both SOD and CAT activity could be interesting against chlorothalonil toxicity, since both enzymes may act as a first line of defense. The decrease, however, observed in the activity of SOD in the digestive gland might indicate an impairment of the antioxidant defense system after the exposure to the biocide. In fact, some studies have demonstrated that this tissue might be an important target for xenobiotic exposure (Faggio et al., 2018). Therefore, the present results suggest that gills and digestive gland have different patterns of responses. When analyzing the basal levels of each component of the antioxidant defense system, gills present higher levels than those observed for the digestive gland. Moreover, both tissues respond differently after exposure to chlorothalonil. Higher GSH levels and increases in SOD activity were observed in gills after the exposure to chlorothalonil. In the digestive gland, lower levels of GSH and decreases in both SOD and GST activities were observed after the exposure to the compound. This might be correlated with the role attributed to the gills in marine mussels, since this is the tissue responsible for the primary internal defenses. Similar patterns of responses are seen for bivalve Scrobicularia plana after exposure to mercury, increasing antioxidant responses in gills and decreasing in digestive gland (Ahmad et al., 2011). Peroxidation levels and protein carbonyls were also analyzed in the present study. In general, the decrease in LPO levels was observed only 6
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Considering our results and those present in the literature (BaierAnderson and Anderson, 2000; Haque et al., 2019), the role of chlorothalonil in causing toxicity to animals and generating oxidative stress can be supported. To mussels P. perna, chlorothalonil, at sub-lethal concentrations, is harmful and might impair their antioxidant and redox status.
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5. Conclusions The exposure of mussels P. perna to chlorothalonil in water can alter their metabolic pathways by changing the antioxidant defense system, which induces an imbalance in the redox state of the animals exposed to this fungicide. Moreover, a tissue-specific response to chlorothalonil was observed. Gills could be acting as the first line of defense, presenting higher enzymatic levels with minor effects on the parameters analyzed. On the other hand, digestive gland, with lower levels of antioxidant defenses, was the most affect organ by chlorothalonil. In this context, it can be suggested that the biocide chlorothalonil is potentially harmful for this species of mussel, being capable of altering glutathione metabolism and other enzymes of the antioxidant defense system. Credit author statement Amanda da Silveira Guerreiro: Conceptualization, Methodology, Formal analysis, Investigation, Writing-Original draft preparation. Fiamma Eugênia Lemos Abreu: Methodology, Investigation, WritingReviewing and Editing. Gilberto Fillmann: Funding acquisition, Conceptualization, Resources, Writing-Reviewing and Editing. Juliana Zomer Sandrini: Conceptualization, Methodology, Resources, Writing Review & Editing, Supervision. Funding This study was sponsored by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES; Finance Code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico – Brazil (CNPq; Proc. No 456,372/2013–0) and FINEP – Pesquisa e Inovação (Proc. No 1111/13–01.14.0141.00). A. S. Guerreiro and F. E. L. Abreu are graduate fellows of CAPES and G. Fillmann is research fellow of CNPq (No 312,341/2013–0 and 314,202/2018–8). Acknowledgments We would like to thank Dr. Regina Coimbra Rola and Heloisa Barbara Gabe for assistance during the experiments. References Ahmad, I., Mohmood, I., Mieiro, C.L., Coelho, J.P., Pacheco, M., Santos, M.A., Duarte, A.C., Pereira, E., 2011. Lipid peroxidation vs. antioxidant modulation in the bivalve Scrobicularia plana in response to environmental mercury — organ specificities and age effect. Aquat. Toxicol. 103, 150–158. Albanis, T.A., Lambropoulou, D.A., Sakkas, V.A., Konstantinou, I.K., 2002. Antifouling paint booster biocide contamination in Greek marine sediments. Chemosphere 48 (5), 475–485. https://doi.org/10.1016/S0045-6535(02)00134-0. Baier-Anderson, C., Anderson, R.S., 2000. The effects of chlorothalonil on oyster hemocyte activation: phagocytosis, reduced pyridine nucleotides, and reactive oxygen species production. Environ. Res. 83, 72–78. https://doi.org/10.1006/enrs.1999. 4033. Bao, V.W.W., Leung, K.M.Y., Qiu, J.-W., Lam, M.H.W., 2011. Acute toxicities of five commonly used antifouling booster biocides to selected subtropical and cosmopolitan marine species. Mar. Pollut. Bull. 62, 1147–1151. https://doi.org/10.1016/j. marpolbul.2011.02.041. Barreto, J.S., Tarouco, F.M., Godoi, F.G.A., Geihs, M.A., Abreu, F.E.L., Fillmann, G., Sandrini, J.Z., da Rosa, C.E., 2018. Induction of oxidative stress by chlorothalonil in the estuarine polychaete Laeonereis acuta. Aquat. Toxicol. (N. Y.) 196, 1–8. https:// doi.org/10.1016/j.aquatox.2017.12.004. Bellas, J., 2006. Comparative toxicity of alternative antifouling biocides on embryos and larvae of marine invertebrates. Sci. Total Environ. 367, 573–585. https://doi.org/10. 1016/j.scitotenv.2006.01.028.
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Effects of chlorothalonil on the antioxidant defense system of mussels Perna perna
T
Amanda da Silveira Guerreiroa,∗, Fiamma Eugênia Lemos Abreub, Gilberto Fillmannb, Juliana Zomer Sandrinia a
Programa de Pós-Graduação em Ciências Fisiológicas, Instituto de Ciências Biológicas, ICB, Universidade Federal do Rio Grande - FURG, 96203-900, Rio Grande, RS, Brazil b Programa de Pós-Graduação em Oceanologia, Instituto de Oceanografia, IO, Universidade Federal do Rio Grande - FURG, 96203-900, Rio Grande, RS, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Antifouling biocides Antioxidant enzymes Bivalve Biomarker Oxidative stress Toxicity
Chlorothalonil is an effective fungicide used in agriculture and formulations of antifouling paints, which use and possible toxicity has been generating great concern. Thus, the present study investigated the effects of chlorothalonil on the antioxidant defense system (ADS) of the mussel Perna perna. The ADS was evaluated in gills and digestive gland after 24 h and 96 h of exposure to environmental relevant levels of chlorothalonil (0.1 and 10 μg/ L). The activity of the enzymes superoxide dismutase (SOD), catalase (CAT), glutamate cysteine-ligase (GCL) and glutathione S-transferase (GST), levels of non-enzymatic defenses, represented by glutathione (GSH), and lipoperoxidation (LPO) and protein carbonyls (PCO) were evaluated. Results indicated that exposure to chlorothalonil is affecting the ADS in both tissues. While the activity of SOD increased and GST and GSH were not altered in gills, they decreased in digestive gland after 24 h of exposure to 10 μg/L of chlorothalonil. The contrasting results indicate that gills and digestive gland presented different patterns of responses after exposure to chlorothalonil. Moreover, a tissue-specific response to chlorothalonil was observed. Gills could be acting as the first line of defense, presenting higher enzymatic levels with minor effects on the parameters analyzed. On the other hand, digestive gland, with lower levels of antioxidant defenses, was the most affect organ by chlorothalonil. It also should be highlighted that the fungicide reduced the glutathione metabolism in the digestive gland, which can lead to an imbalance of the redox state within the cells of animals.
1. Introduction
commonly used since they are considered less toxic and less persistent in the environment (Konstantinou and Albanis, 2004; Thomas and Brooks, 2010; Voulvoulis et al., 2002). Chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile) is an aromatic halogen compound that was first introduced as a broad-spectrum fungicide in agriculture (Ernst et al., 1991). Due to its antifungal properties, it was also introduced as a booster biocide in commercial antifouling paints as well. Environmental levels ranging from 0.004 to 1.38 μg/L were already reported for marine and freshwater ecosystems, demonstrating its dual usage (Edwards et al., 2017; Lee et al., 2010; Voulvoulis et al., 2000). In the Korean coast, Lee et al. (2010, 2015) reported concentrations close to 0.067 μg/L in water and 50.6 ng/L in sediment samples and associated those values with the increased activity of the harbors nearby. Levels of the contaminant were also found in rivers, which is correlated with the agricultural activity of close areas (Callicott and Hooper-Bùi, 2019). Those levels were ranging from 0.0035 μg/L to 0.024 μg/L and were observed in superficial waters of
Marine biofouling is a natural phenomenon that often poses a problem for the shipping industry. Biofouling, defined as the accumulation of microorganisms, plants, and aquatic invertebrates on surfaces immersed in seawater, can increase the roughness and frictional resistance of ships, leading to an increase in fuel consumption and docking frequency (Omae, 2003; Yebra et al., 2004). To prevent the settlement and growth of organisms on those surfaces, antifouling paints have been developed and used extensively. Organotin-based compounds, such as tributyltin (TBT), were the most successful against fouling (Omae, 2003). However, these compounds were banned due to their persistence and toxicity (Bao et al., 2011; Bigatti et al., 2009; Evans et al., 2000; Ofoegbu et al., 2016; Strand et al., 2009). In this context, safer alternatives to TBT have been developed and introduced in paint formulations. Booster biocides, such as copper-pyrithione, zinc-pyrithione, DCOIT, Irgarol 1051, diuron, and chlorothalonil, are some of the most
∗
Corresponding author. E-mail address: [email protected] (A.d.S. Guerreiro).
https://doi.org/10.1016/j.ecoenv.2019.110119 Received 11 September 2019; Received in revised form 16 December 2019; Accepted 20 December 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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every two days and then, water was renewed.
the Mississippi river (Callicott and Hooper-Bùi, 2019). Information concerning chlorothalonil contamination levels in the coast of Brazil and other Latin American countries is still not available. Some studies have demonstrated that chlorothalonil can be harmful to the aquatic biota, inducing DNA damage (Cima et al., 2008), and causing growth abnormalities (Bellas, 2006) and mortality in different marine species (Ernst et al., 1991; Key et al., 2003; Koutsaftis and Aoyama, 2007). For bivalves, a 96 h EC50 of 8.8 μg/L (Bellas, 2006) and a 96 h LC50 of 5900 μg/L (Ernst et al., 1991) was reported for larvae and adults of Mytilus edulis, respectively, exposed to chlorothalonil or exposed to a formulated product (BRAVO 500) containing chlorothalonil. In the ascidian Bothryllus schollosseri, exposure to chlorothalonil resulted in loss of function of mitochondria, lower ATP production, and subsequent apoptosis (Cima et al., 2008). In hemocytes of the mussel Perna perna, exposure to chlorothalonil caused decrease in cellular viability and increase in cellular adhesion and phagocytic activity (Guerreiro et al., 2017). It has been proposed that this biocide has the ability to bind to sulfhydryl groups of peptides and proteins, which could lead to effects like reduction of glutathione levels and inhibition of enzymes (i.e. NADPH oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) (Baier-Anderson and Anderson, 2000; Tillman et al., 1973; Long and Siegel, 1975). Indeed, some authors (Davies, 1985; Gallagher et al., 1991) have demonstrated that the tripeptide glutathione (GSH) and the enzyme glutathione S-transferase (GST) are important in chlorothalonil metabolism and toxicity. They observed the formation of chlorothalonil-glutathione conjugates in fish species and suggested that GST has an important role in chlorothalonil toxicity and detoxification. Considering that GSH and GST are important in the biotransformation process, as well as in the antioxidant defense system, studies have proposed that, similar to many other environmental pollutants, chlorothalonil could induce oxidative stress by altering the balance between those molecules (Cima et al., 2008; Tillman et al., 1973; Baier-Anderson and Anderson, 2000). Recently, Barreto et al., (2018) observed that chlorothalonil induced oxidative stress in the estuarine polychaeta Laeonereis acuta through reduction of its total antioxidant capacity and alteration in glutathione metabolism, observed as altered activities of glutamate cysteine-ligase and glutathione S-transferase. In this context, chlorothalonil might impair the antioxidant defense systems of other organisms by changing the redox status of cells. Seeing the hazard of chlorothalonil exposure to aquatic organisms, the present study aimed to investigate its effects on the antioxidant defense system of the mussel P. perna. Mussels have been widely used in biomonitoring programs (Goldberg, 1986; Goldberg and Bertine, 2000) and species, such as P. perna, are widely distributed along the Brazilian coast (Siddall, 1980). In addition, mussels allow an assessment of environmental contaminants partitioned either in the dissolved (by respiration) and particulate (by filter-feeding) phases of water column (Dame, 1996). In this context, the antioxidant defense system of P. perna was investigated after exposure to the biocide chlorothalonil. The concentrations tested in the present study (0.1 μg/L and 10 μg/L) are environmentally relevant and considered sublethal to P. perna.
2.2. Experimental design Mussels (N = 72) were separated into nine 6-L tanks to perform the experiment in triplicates (three aquaria for each treatment). The water conditions described previously were maintained until the end of the experimental period. Animals were exposed to the biocide chlorothalonil for up to 96 h at the following nominal concentrations: control (with 0.01% dimethyl sulfoxide), and 0.1 μg/L (0.37 nM) and 10 μg/L (37 nM) of chlorothalonil. After 24 h and 96 h of exposure, 6 mussels of each treatment (2 of each aquaria) were dissected, and gills and digestive gland were collected. Tissues were stored at −80 °C until further analyses. Concentrations of chlorothalonil in water were measured as described by Barreto et al. (2018). Briefly, water samples were extracted by SPE (C18 cartridge) and the eluates analyzed by gas chromatography/electron capture detector (GC-ECD). Measurements were conducted immediately after contaminant addition (0 h) and after 48 h of exposure. 2.3. Biochemical biomarkers For enzymatic measures of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) activity, mussel tissues (gills and digestive gland) were homogenized (1:4 w/v) in cold buffer (20 mM Tris base, 1 mM EDTA, 0.5 M sucrose, 0.15 M KCl, and 0.1 mM PMSF, pH 7.6). Samples were centrifuged at 1000 g for 20 min at 4 °C and subsequently at 10,000 g for 45 min at 4 °C. Supernatant was collected while the resulting pellet was discarded. Protein concentration was evaluated by the biuret method. SOD activity was measured in a spectrophotometer at 550 nm according to McCord and Fridovich (1969). In this assay, the reduction of cytochrome C was evaluated through the inhibition of xanthine:xanthine oxidase. One SOD unit represents the amount of enzyme necessary for 50% inhibition of cytochrome C at 25 °C. CAT activity was evaluated through the decomposition of H2O2 per minute at 240 nm, as described by Beutler (1975), and was expressed as CAT units. One CAT unit represents the amount of enzyme able to decompose 1 μmol of H2O2 per minute per milligram of protein. GST activity was measured at 340 nm, using 1-chloro-2,4-dinitro- benzene (CDNB, Sigma) as a substrate for its activity (Habig et al., 1974). The results were expressed as GST units, where one GST unit represents the amount of enzyme necessary to conjugate 1 μmol of CDNB per minute per milligram of protein at 25 °C. Considering glutathione metabolism, glutamate cysteine-ligase (GCL) activity and glutathione (GSH) content were measured following the protocol of White et al. (2003). Mussel tissues (gills and digestive gland) were first homogenized (1:5 w/v) in cold buffer (100 mM TrisHCl, 2 mM EDTA, 5 mM MgCl2.6H2O) and centrifuged at 20,000 g for 20 min at 4 °C. The pellet was then discarded, and the supernatant was stored at −80 °C. Extracts were then evaluated following the reaction of the fluorescent compound 2,3-naphtalenedicarboxaldehyde (NDA) with GSH and with γ-glutamyl-cysteine residues. The reaction product was analyzed in a fluorimeter (Victor, PerkinElmer) at 472 nm (excitation) and 528 nm (emission). The GCL activity was expressed as ηmol of GSH per hour and per mg of protein, while the GSH concentration was expressed as mg of GSH per mg of protein.
2. Materials and methods 2.1. Animals P. perna were obtained from a mariculture farm located in the southwest of Santa Catarina Island, Brazil (−27.729,769, −48.562,973). Animals were transported to the Universidade Federal do Rio Grande – FURG, where the experiments were conducted. Thereafter, mussels were acclimated for 15 days in tanks (20 L) with filtered (5 μm) seawater (salinity of 30), under constant aeration, temperature of 20 ± 2 °C, and photoperiod of 12L:12D. Animals were fed with phytoplankton Nannochloropsis sp and Conticribra weisfloggii in
2.4. Quantification of lipoperoxidation levels (LPO) Lipid peroxidation was evaluated through the TBARS assay, according to Oakes and Van der Kraak, (2003). The end products of lipid peroxidation were measured considering the reaction of substances, mainly malondialdehyde, with thiobarbituric acid. In this assay, samples were homogenized (1:5 w/v) in cold buffer (100 mM Tris-HCl, 2
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0.1 μg/L and 10 μg/L of chlorothalonil for 24 h (Fig. 3A), but no effect was observed after 96 h of exposure. No alterations were observed in the digestive gland (Fig. 3B). Although no effects were observed in gills (Fig. 3C), a decrease in levels of protein carbonyls was detected in digestive glands exposed to 10 μg/L of chlorothalonil for 24 h and 96 h (Fig. 3D).
2 mM EDTA, 5 mM MgCl2.6H2O) and centrifuged at 20,000 g for 20 min at 4 °C. The supernatant was incubated with thiobarbituric acid, butylated hydroxytoluene, acetic acid and 8.1% SDS buffer and, then, heated to 95 °C for 30 min to promote a color reaction. Analyses were carried out in a fluorimeter (Victor, PerkinElmer) at 515 nm (excitation) and 553 nm (emission). The results were expressed as nmol MDA per mg protein.
4. Discussion 2.5. Protein carbonyl content (PCO) Despite the economic importance to naval industry, it is known that biocides presented in antifouling paints may also cause toxicity to nontarget species. Diuron and Irgarol, for example, are considered extremely harmful to marine phytoplankton (Bao et al., 2011; DeLorenzo and Fulton, 2012). Both biocides are known photosynthetic system II inhibitors, and can affect cyanobacteria, algae and the symbiotic dinoflagellates presented in corals (Jones and Kerswell, 2003). DCOIT also has effects on algae development and growth (Onduka et al., 2013), and studies clearly demonstrates its toxic potential to fish and polychaetas as well (Chen et al., 2016; Onduka et al., 2013). Chlorothalonil, on the other hand, is a fungicide widely applied in agricultural sites and as a co-biocide in antifouling paints that still lacks information concerning its toxic effects on terrestrial and aquatic species. A previous study conducted by our group had already demonstrated sublethal effects in the immune system of mussel P. perna after exposure to 0.1 μg/L and 10 μg/L of chlorothalonil (Guerreiro et al., 2017). Those concentrations might be representative of the actual scenario in many aquatic ecosystems, since similar values have already been reported worldwide. Lee et al. (2010) and Sakkas et al., 2002b, for example, have found chlorothalonil levels as high as 0.07 μg/L and 0.06 μg/L in water of harbor areas from Korea and Greece, respectively. Other studies reported even higher concentrations (1.38 μg/L) in estuarine areas of the UK, which correlates with the end of local boating season (Voulvoulis et al., 2000). Chlorothalonil was also reported in sediments at concentrations up to 690 ng/g, impacting both nektonic and benthic organisms (Albanis et al., 2002; Lee et al., 2015). Although many studies have been detecting chlorothalonil in water and sediments, this compound appears to present a relatively rapid degradation and/or metabolization in the environment (Davies, 1985; Gallagher et al., 1991; Sakkas et al., 2002a). Since chlorothalonil is expected to degrade within 48 h in natural seawater (Sakkas et al., 2002a), water treatments were renewed every two days during the present study. Even so, measured chlorothalonil levels in water were < LOD at all treatments after 48 h which might be due to the uptake and/or metabolization by mussels. As filter-feeding organisms, mussels not only remove materials from the water column, but they can induce nutrient cycling in the environment (Dame, 1996). Also, they are capable of accumulating many types of contaminants in their soft tissues (Sehonova et al., 2018; Viarengo and Canesi, 1991), such as metals (Estrada et al., 2017), hydrocarbons (Lüchmann et al., 2011), pesticides (Pariseau et al., 2009), and other organic compounds (Varol and Sünbül, 2017; Quintas et al., 2017), which can induce several kinds of damage related to contaminant exposure and accumulation. Results from Davies and White (1985) suggest that chlorothalonil can accumulate in fish tissues. However, for bivalve species, such as the mussel P. perna, information regarding chlorothalonil accumulation and metabolization still need a thorough assessment. In any case, accumulating or not, exposure to chlorothalonil causes several types of damage to organisms (Barreto et al., 2018; Cima et al., 2008), including to the immune system of mussels (Guerreiro et al., 2017). Some studies have demonstrated that biotransformation of chlorothalonil in marine and terrestrial organisms is mainly through the conjugation with glutathione (Kim et al., 2004; Rosner et al., 1996). In rats, Rosner et al., (1996) observed that chlorothalonil was rapidly transformed to 4,6-bis(glutathion-S-yl)-2,5-dichloroisophthalonitril in
Protein carbonyl content was determined using the OxySelect™ Protein Carbonyl Fluorimetric Assay Kit (MyBioSource). Briefly, samples were homogenized (1:4 w/v) in cold buffer (20 mM Tris base, 1 mM EDTA, 0.5 M sucrose, 0.15 M KCl, and 0.1 mM PMSF, pH 7.6) and centrifuged twice (1000 g for 20 min at 4 °C and subsequently at 10,000 g for 45 min at 4 °C). Supernatants were then incubated with the OxiSelect™ fluorophore. After the incubation period, 20% trichloroacetic acid solution was added to induce the precipitation of proteins. Free fluorophore was then removed by acetone washes. Absorbance was measured fluorometrically with a 485/538 nm filter. The results were expressed as nmol per mg of protein. 2.6. Statistical analysis Results are presented as mean ± standard error. Data were tested for normality and homoscedasticity and, when complied with the assumption, one-way ANOVA followed by Tukey HSD post hoc test were applied. For nonparametric data, Kruskal-Wallis test followed by the post-hoc of multiple comparisons of the mean ranks were applied for all groups. Statistical analyses were performed using Statistic 7 (StatSoft) with a significance level of 5% (p < 0.05). 3. Results Despite no mortality was observed, exposure to both concentrations of chlorothalonil (0.1 μg/L and 10 μg/L) altered the parameters assessed of the antioxidant defense system of mussels. Chlorothalonil concentrations evaluated in each treatment were 0.1 μg/L (nominal concentration of 0.1 μg/L) and 6.5 μg/L (nominal concentration of 10 μg/L) at 0 h. For the control treatment, the measured chlorothalonil concentration was lower than the limit of detection (LD < 0.1 μg/L). Water samples collected at 48 h were also analyzed to quantify chlorothalonil at the end of treatments. For each treatment, including the control group, the measured chlorothalonil concentration was lower than the limit of detection (LD < 0.1 μg/L). Concerning SOD activity, an increase was observed in gills exposed to the highest concentration of chlorothalonil (10 μg/L) for 24 h. This effect was not observed after 96 h of exposure (Fig. 1A). Despite this increase in gills, a decrease in its activity was observed in digestive gland exposed to both concentrations tested for 24 h. Again, this response was not observed at the end of the experimental period (96 h) (Fig. 1B). The activity of catalase was not significantly altered by any of the concentrations tested in gills (Fig. 1C) or digestive gland (Fig. 1D) during either of the experimental periods. Regarding GST activity, no effects of chlorothalonil were observed in gills after 24 h or 96 h of exposure (Fig. 1E). However, a decrease in the activity of this enzyme was observed in digestive gland exposed to both treatments for 24 h. This effect was not observed after 96 h of exposure (Fig. 1F). A decrease in GCL activity was observed in gills of animals exposed to 10 μg/L for 24 h and to 0.1 μg/L and 10 μg/L of chlorothalonil for 96 h (Fig. 2A). In the digestive gland, an increase in GCL activity was observed in animals exposed to 0.1 μg/L of chlorothalonil for 96 h (Fig. 2B). On contrary, GSH levels were not significantly altered in the gills (Fig. 2C), while a reduction in levels of glutathione was observed in digestive gland exposure to 10 μg/L of the biocide for 24 h (Fig. 2D). Levels of lipid peroxidation (as TBARS) reduced in gills exposed to 3
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Fig. 1. Activity (mean ± standard error, N = 72) of superoxide dismutase (SOD) (A and B), catalase (CAT) (C and D), and glutathione S-transferase (GST) (E and F) in gills (left) and digestive gland (right) of mussels P. perna exposed to 0.1 μg/L and 10 μg/L of chlorothalonil for 24 h and 96 h. Different letters indicate statistically significant differences (p < 0.05).
of NADPH oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has been discussed (Long and Siegel, 1975; Baier-Anderson and Anderson, 2000). Glutathione (GSH), a known target of chlorothalonil, is a tripeptide that contains a sulfhydryl group due to the presence of a cysteine residue and participates in the antioxidant defense system of organisms in order to maintain the redox state of cells (Regoli and Giuliani, 2014). Moreover, it is an important agent in cellular detoxification processes, since it can be conjugated with endogenous or exogenous compounds (Meister and Anderson, 1983). In this context, GSH can intercept electrophilic and oxidant species, preventing damage to nucleic acids and proteins (Pompella et al., 2003; Trevisan et al., 2016). Elevated GSH levels are usually related to an
the presence of glutathione in the rat liver cytosol. Mono-glutathione conjugates are observed as intermediates in the enzymatic reaction, while tri-glutathione conjugates are the final metabolite in the biotransformation of chlorothalonil (Kim et al., 2004). This might indicate that the toxicity of the compound is related with its own biotransformation process and that might be related with the altered antioxidant pathways. The underlying mechanisms involved in chlorothalonil toxicity are still under investigation. Nevertheless, studies have already demonstrated that this compound has binding affinity for the sulfhydryl groups of peptides and proteins (Tillman et al., 1973). Its involvement in cellular respiration and in the glycolytic pathway, through inhibition 4
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Fig. 2. Glutamate cysteine-ligase (GCL) activity (A and B) and glutathione (GSH) (C and D) content (mean ± standard error, N = 72) in gills (left) and digestive gland (right) of mussels P. perna exposed to 0.1 μg/L and 10 μg/L of chlorothalonil for 24 h and 96 h. Different letters indicate statistically significant differences (p < 0.05).
would be important for the protection of organisms against chlorothalonil toxicity, since this compound is mainly metabolized through glutathione conjugates (Gallagher et al., 1991; Rosner et al., 1996). The present results, however, suggest that decreases in GST activity might be correlated with an increase in biotransformation processes and/or the inability of the animal in producing enough GST to cope with the contaminant exposure, at least in the period evaluated. As observed by Gallagher et al., (1991) and by Rosner et al., (1996), GST involvement in chlorothalonil biotransformation is clear, therefore, its decreased activity may be causing toxicity to the animals. Both SOD and CAT also participate in the antioxidant defense system of organisms, protecting cells from oxidative damage caused by the exposure to contaminants (Livingstone, 2001). According to Escobar et al., (1996), alterations in SOD activity might even be related to production of reactive oxygen species (ROS). Some authors suggest that elevated ROS levels may inhibit some antioxidant enzymes, such as SOD (Escobar et al., 1996), while others suggest that the increase in ROS production might be the signal to drive antioxidant responses (Zhao et al., 2017). Although not evaluated in the present study, ROS levels might impact the enzymes involved in the antioxidant defense system, as previously commented. SOD activity was increased in the gills and decreased in the digestive gland after 96 h of exposure to chlorothalonil, and such a difference may be related to organ specificity. Considering that gills are the major site of uptake of chemicals from water (Hayton and Barron, 1990), the increase observed in SOD activity
increase in capacity to resist oxidative stress, while reduced levels might lead to an increase in oxidative damage (Faggio et al., 2016; Gobi et al., 2018). As observed by Davies, (1985) and Gallagher et al., (1991), chlorothalonil targets GSH and promotes a marked decrease of the tripeptide level. However, the decrease in GSH in fish is followed by an increase in GST activity, leading to the formation of mono-, di-, and tri-glutathione/chlorothalonil conjugates. In the present study, alterations in GSH levels were observed in digestive gland only. Although no effects were observed in gills, levels of GSH were at least 3 times higher than in digestive gland. These decreases observed in digestive gland might be related to chlorothalonil's biotransformation within the tissues. Considering that gills are key players in bivalve defenses, they can act as a metabolic barrier against an electrophilic burden, thereby proving a stronger defense against many contaminants (Ahmad et al., 2011; Trevisan et al., 2014, 2016). In addition to GSH, chlorothalonil was responsible for altering levels of other enzymes related to glutathione metabolism. The activity of GCL, the main enzyme responsible for GSH biosynthesis through the binding of glutamic acid and cysteine (Regoli and Giuliani, 2014), was decreased in the gills after 24 h and 96 h of exposure. Despite that, this decrease does not seem to interfere with the GSH levels observed. An increase in GCL activity, however, was observed only in polychaetas exposed to 100 μg/L of chlorothalonil for 24 h (Barreto et al., 2018), which was 10 times higher than those used in the present study. It is known that under different environmental stress situations, GSTs can be altered (van der Oost et al., 2003). Increases in GST activity 5
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Fig. 3. Lipoperoxidation (as TBARS) (A and B) and protein carbonyl (C and D) levels (mean ± standard error, N = 72) in gills (left) and digestive gland (right) of mussels P. perna exposed to 0.1 μg/L and 10 μg/L of chlorothalonil for 24 h and 96 h. Different letters indicate statistically significant differences (p < 0.05).
in gills and may be correlated with higher basal levels of most of the enzymes analyzed. As reported by Baier-Anderson and Anderson (2000), a suppression in superoxide production, evaluated indirectly by the increase in the activity of SOD, may be an explanation for the decrease in the damage of gills. For the PCO results, a decrease in digestive gland of the organisms exposed to chlorothalonil was observed. Since chlorothalonil targets the antioxidant defenses of organisms, it can be suggested that the main antioxidant pathway, driven by the Nrf 2 (nuclear factor erythroid 2 – related factor 2), is modulating the activation of protective agents, such as α-tocopherol. Indeed, Guerreiro and colleagues (manuscript submitted to publication) showed that chlorothalonil affects most of AhR and Nrf2-targeted genes, such as GSTs, SOD and CYP1A2, including AhR own expression. Therefore, the toxicity of chlorothalonil can be correlated with alterations in the antioxidant pathways, which are extremely important for avoiding oxidative stress situations. It is important to highlight that basal levels of enzymatic activity, as well as levels of PCO, were quite different among the tissues analyzed. In general, digestive gland was the most impaired tissue. Basal levels regarding GSH and PCO content demonstrated that higher oxidation in proteins and lesser defenses are frequently observed in digestive gland. Gills, on the other hand, present higher levels of GSH and other antioxidant enzymes. These results corroborate with the role of gills as a metabolic barrier against xenobiotic exposure, and of digestive gland as a more vulnerable tissue in front of exposure to toxicants. This vulnerability makes this tissue important in ecotoxicology analyses, as evaluated by Faggio et al., (2018).
at this tissue may be important since indicate a better protection against ROS formation. Haque et al., (2019) also demonstrates that an increase in the activity of this enzyme is expected for other bivalve species, such as Crassostrea gigas and Mytilus edulis. Those authors reported that increases in both SOD and CAT activity could be interesting against chlorothalonil toxicity, since both enzymes may act as a first line of defense. The decrease, however, observed in the activity of SOD in the digestive gland might indicate an impairment of the antioxidant defense system after the exposure to the biocide. In fact, some studies have demonstrated that this tissue might be an important target for xenobiotic exposure (Faggio et al., 2018). Therefore, the present results suggest that gills and digestive gland have different patterns of responses. When analyzing the basal levels of each component of the antioxidant defense system, gills present higher levels than those observed for the digestive gland. Moreover, both tissues respond differently after exposure to chlorothalonil. Higher GSH levels and increases in SOD activity were observed in gills after the exposure to chlorothalonil. In the digestive gland, lower levels of GSH and decreases in both SOD and GST activities were observed after the exposure to the compound. This might be correlated with the role attributed to the gills in marine mussels, since this is the tissue responsible for the primary internal defenses. Similar patterns of responses are seen for bivalve Scrobicularia plana after exposure to mercury, increasing antioxidant responses in gills and decreasing in digestive gland (Ahmad et al., 2011). Peroxidation levels and protein carbonyls were also analyzed in the present study. In general, the decrease in LPO levels was observed only 6
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Considering our results and those present in the literature (BaierAnderson and Anderson, 2000; Haque et al., 2019), the role of chlorothalonil in causing toxicity to animals and generating oxidative stress can be supported. To mussels P. perna, chlorothalonil, at sub-lethal concentrations, is harmful and might impair their antioxidant and redox status.
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5. Conclusions The exposure of mussels P. perna to chlorothalonil in water can alter their metabolic pathways by changing the antioxidant defense system, which induces an imbalance in the redox state of the animals exposed to this fungicide. Moreover, a tissue-specific response to chlorothalonil was observed. Gills could be acting as the first line of defense, presenting higher enzymatic levels with minor effects on the parameters analyzed. On the other hand, digestive gland, with lower levels of antioxidant defenses, was the most affect organ by chlorothalonil. In this context, it can be suggested that the biocide chlorothalonil is potentially harmful for this species of mussel, being capable of altering glutathione metabolism and other enzymes of the antioxidant defense system. Credit author statement Amanda da Silveira Guerreiro: Conceptualization, Methodology, Formal analysis, Investigation, Writing-Original draft preparation. Fiamma Eugênia Lemos Abreu: Methodology, Investigation, WritingReviewing and Editing. Gilberto Fillmann: Funding acquisition, Conceptualization, Resources, Writing-Reviewing and Editing. Juliana Zomer Sandrini: Conceptualization, Methodology, Resources, Writing Review & Editing, Supervision. Funding This study was sponsored by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES; Finance Code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico – Brazil (CNPq; Proc. No 456,372/2013–0) and FINEP – Pesquisa e Inovação (Proc. No 1111/13–01.14.0141.00). A. S. Guerreiro and F. E. L. Abreu are graduate fellows of CAPES and G. Fillmann is research fellow of CNPq (No 312,341/2013–0 and 314,202/2018–8). Acknowledgments We would like to thank Dr. Regina Coimbra Rola and Heloisa Barbara Gabe for assistance during the experiments. References Ahmad, I., Mohmood, I., Mieiro, C.L., Coelho, J.P., Pacheco, M., Santos, M.A., Duarte, A.C., Pereira, E., 2011. Lipid peroxidation vs. antioxidant modulation in the bivalve Scrobicularia plana in response to environmental mercury — organ specificities and age effect. Aquat. Toxicol. 103, 150–158. Albanis, T.A., Lambropoulou, D.A., Sakkas, V.A., Konstantinou, I.K., 2002. Antifouling paint booster biocide contamination in Greek marine sediments. Chemosphere 48 (5), 475–485. https://doi.org/10.1016/S0045-6535(02)00134-0. Baier-Anderson, C., Anderson, R.S., 2000. The effects of chlorothalonil on oyster hemocyte activation: phagocytosis, reduced pyridine nucleotides, and reactive oxygen species production. Environ. Res. 83, 72–78. https://doi.org/10.1006/enrs.1999. 4033. Bao, V.W.W., Leung, K.M.Y., Qiu, J.-W., Lam, M.H.W., 2011. Acute toxicities of five commonly used antifouling booster biocides to selected subtropical and cosmopolitan marine species. Mar. Pollut. Bull. 62, 1147–1151. https://doi.org/10.1016/j. marpolbul.2011.02.041. Barreto, J.S., Tarouco, F.M., Godoi, F.G.A., Geihs, M.A., Abreu, F.E.L., Fillmann, G., Sandrini, J.Z., da Rosa, C.E., 2018. Induction of oxidative stress by chlorothalonil in the estuarine polychaete Laeonereis acuta. Aquat. Toxicol. (N. Y.) 196, 1–8. https:// doi.org/10.1016/j.aquatox.2017.12.004. Bellas, J., 2006. Comparative toxicity of alternative antifouling biocides on embryos and larvae of marine invertebrates. Sci. Total Environ. 367, 573–585. https://doi.org/10. 1016/j.scitotenv.2006.01.028.
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