Effects of selenium treatment on endogenous antioxidant capacity in signal crayfish (Pacifastacus leniusculus)

Abstract

Selenium is an essential element that plays a role in numerous physiological processes and is critical for the maintenance of a strong endogenous antioxidant system. Previous work by our research group reported that the organophosphate pesticide dimethoate decreased glutathione S-transferase activity (GST) in signal crayfish (Pacifastacus leniusculus) collected from the Boise River (Idaho, USA). The goals of this study were to examine whether: 1) sodium selenite modulated the endogenous antioxidants glutathione (GSH), metallothionein (MT), and glutathione S-transferase (GST), thus suggesting a mechanism of antioxidant activity, 2) dimethoate exposure (pro-oxidant stressor) decreased GST activity in a localized population of signal crayfish collected from the Snake River (Idaho, USA), and 3) investigate whether selenium cotreatment ameliorated the adverse effects of dimethoate on GST activity due to the antioxidant properties associated with selenium. Selenium and dimethoate treatments (and co-treatments) did not modulate GSH or MT concentrations at the doses tested in this study. Furthermore, neither selenium nor dimethoate were factors influencing GST activity, and no interaction was found between the treatments. While our results did not support our predictions, they are suggestive and future studies examining the protective role of selenium in pro-oxidant exposure in this species are warranted. Population-specific responses as well as seasonal variations in endogenous antioxidant expression should be considered in future experiments.

Graphical Abstract

1. Introduction

Selenium is an essential element that plays a critical role in growth, immune function, reproduction, and support of endogenous antioxidant systems, although adverse effects can result from high levels of exposure (Chen et al., 2020; Elia et al., 2011; Mechlaoui et al., 2019; Mo et al., 2019; Nugroho and Fotedar, 2013; Wang et al., 2019). Selenium bioaccumulates in food webs and can lead to toxic exposure in organisms inhabiting impacted ecosystems (Beckon, 2016; Johnson et al., 2009). Selenium can enter the environment through sources that include agricultural runoff and coal ash, the byproduct of coal combustion (Johnson et al., 2009; Otter et al., 2012). It is also a common supplement used to enhance the nutritional quality of aquaculture feed (Dorr et al., 2008). In crayfish (Procambarus clarkii), selenium has been associated with enhanced growth rates and reductions in health indicator values, an effect that is thought to be due to depletion of energy stores in the hepatopancreas (Doerr et al., 2013). Furthermore, selenium treatment in female crayfish (Procambarus clarkii) improved spawning rates, promoted synchronized spawning, and increased hemolymph E₂ and vitellogenin concentrations, although adverse effects were observed at higher concentrations with decreases in spawning rates and E₂ concentrations (Mo et al., 2019). In another crayfish species, Cherax cainii, selenium increased immune function, growth, and survival, and increased resistance to bacteria infection by Vibrio mimicus (Nugroho and Fotedar, 2013). Selenium also plays a critical role it the maintenance and support of a strong endogenous antioxidant system. It is involved in the the expression of 25 selenoproteins and effects non-enzymatic antioxidants such as vitamin E, CoQ, and GSH (Surai and Kochish, 2019). Given the antioxidant properties associated with selenium, we were interested in examining whether it regulates endogenous antioxidant and detoxification pathways in signal crayfish (Pacifastacus leniusculus).

Signal crayfish are endemic to the Pacific Northwest of North America and they have been introduced into other regions of North America, Europe, and Japan (Holdich, 2002; Larson and Olden, 2011). Introductions into new locations were often driven by interests in commercially farming the species, introducing it as a prey base for game fish, replacing crayfish plague sensitive species, or use as bait and subsequent release leading to the establishment of stable populations (Holdich, 2002). Aquaculture has proven difficult in this species given its relatively slow growth rates and tendency for cannibalism (Holdich, 2002). Crayfish are considered keystone species and important links in the energy flow within and between terrestrial and aquatic systems given their omnivorous diet (plant and animal matter, detritus, etc.) and role as a food source for both aquatic (fish) and terrestrial (racoons, birds, etc.) predators (Larson and Olden, 2011). Signal crayfish inhabit relatively localized home ranges and bioaccumulate contaminants making them excellent model organisms for use in ecotoxicology studies (Anton et al., 2000; Beganyi and Batzer, 2011; Bennet-Chambers et al., 1999; Bowling et al., 2011; Faria et al., 2010; Gentes et al., 2013; Guan and Wiles, 1997; Kouba et al., 2010; Mueller and Serdar, 2002; Suarez-Serrano et al., 2010).

Glutathione S-transferases (GST), glutathione (GSH), and metallothioneins (MT) play important roles in detoxification, neutralizing free radicals associated with oxidative stress, and the sequestration, detoxification, and excretion of essential (Zn, Cu) and non-essential (Hg, Cd, etc.) metals (Amiard et al., 2006; Dickinson and Forman, 2002; Newman, 1998; Park et al., 2020). Selenium treatment is known to modulate these pathways and, in some cases, ameliorate the adverse effects of pro-oxidant exposure. For example, red swamp crayfish (Procambarus clarkii) treated with selenium demonstrated lower GSH concentrations and changes in GST (Dorr et al., 2008). Furthermore, increases in GSH in response to selenium have been reported in Oriental river prawn (Macrobrachium nipponense) and blue mussels (Mytilus edulis) (Kong et al., 2017; Trevisan et al., 2011). Tilapia (Oreochromis mozambicus) exposed to selenium exhibited increased GST activity in gill, decreased GST activity in liver, and increases in both MT and GSH in gill and liver tissues (Gobi et al., 2018). Based on these studies, selenium has the potential to modulate antioxidant defense mechanisms and could thus elicit a protective effect against oxidative damage caused by pro-oxidants. This is supported by studies demonstrating that selenium treatment protects against oxidative damage caused by pro-oxidants that include aluminum chloride (rat), copper sulfate (blue mussels – Mytilus edulis), methyl parathion (fish – Brycon cephalus), and dimethoate (rat) in a range of taxa (Ben Amara et al., 2011a; Ben Amara et al., 2011b; Ghorbel et al., 2017; Monteiro et al., 2009; Trevisan et al., 2011).

Dimethoate is an organophosphate pesticide commonly used in agriculture and has been shown to modulate markers indicative of oxidative stress as well as endogenous antioxidant pathways in rats (Ben Amara et al., 2011a; Ben Amara et al., 2011b; Sharma et al., 2005a; Sharma et al., 2005b). The collective effects of Cytochrome P-450 induction, modulations in GSH and GST, and the inhibition of acetylcholinesterase (AChE) are thought to be the root cause of the oxidative stress generated by dimethoate (Sharma et al., 2005b). Dimethoate exposure leads to increases is lipid peroxidation (LPO), decreases in GSH, inhibition of AChE and Na⁺-K⁺-ATPase, and depletion of vitamin C in rats (Ben Amara et al., 2011a; Sharma et al., 2005b). Interestingly, treatment with selenium (and/or vitamin E) diminishes the effects of dimethoate, presumably through mechanisms related to antioxidant defense (Ben Amara et al., 2011a; Ben Amara et al., 2011b).

In this study, we examined the regulation of the endogenous antioxidants GST, GSH, and MT by selenium alone, as well as in combination with dimethoate (pro-oxidant stressor), in signal crayfish to determine whether it regulates these pathways and could potentially protect against dimethoate induced decreases in GST activity previously reported in this species (Gunderson et al., 2018). We predicted that selenium alone would modulate these pathways and furthermore ameliorate the inhibition of GST by dimethoate when administered as a cotreatment.

2. Methods

2.1. Animal collection, exposures, and colorimetric assays

Crayfish (body length (rostrum to tail tip) 101.3 +/− 1.4 mm; body weight 40.0 +/− 1.2 g (mean +/− S.E)) were collected from C.J. Strike Reservoir (N 42.95148° W 115.97243°) on the Snake River (Idaho, USA) using baited traps during October - November 2017 under Idaho Department of Fish and Game permit # F-12–04-17. Animals were housed and injected with the respective treatments as described in earlier studies conducted by our research group (Gunderson et al., 2021; Gunderson et al., 2018). Briefly, animals were maintained in aerated tanks containing dechlorinated tap water at room temperature, on a light cycle approximating early summer in southwestern Idaho (~6:30 – 22:30). Crayfish were injected with vehicle (400 mM NaCl), dimethoate (0.9 µg g⁻¹), sodium selenite (0.1, 0.5, 1.0 µg g⁻¹), or co-injected with dimethoate (0.9 µg g⁻¹) and sodium selenite (0.1, 0.5, 1.0 µg g⁻¹). Selenium concentrations were roughly in the range of selenium accumulated in the hepatopancreas (~0.25–0.7 µg g⁻¹) of red swamp crayfish (Procambarus clarkii) fed selenium enriched diets (0.30 and 1.21 µg g⁻¹) for 50 days (Dorr et al., 2008). The dimethoate concentration chosen was based on a previous study by our research group demonstrating that GST activity was inhibited in signal crayfish injected with dimethoate at concentrations of 0.3, 0.6, and 0.9 µg kg⁻¹ (Gunderson et al., 2018). A higher dose (0.9 µg g⁻¹ dimethoate) was chosen for this study with the assumption that it would provide a more potent effect. Each crayfish was placed in an aerated 7L glass fishbowl containing deionized water supplemented with Instant Ocean™ (200 mg L⁻¹) and water was changed every 24 h. After 72 h, animals were chilled in ice water and sacrificed by decapitation. Hepatopancreas tissue was removed, flash frozen in liquid nitrogen, and stored at −80 °C until processed.

2.2. Glutathione Concentration (GSH)

GSH concentrations were measured using a previously published DTNB (5,5’ -dithiobis(nitrobenzoic acid)) based colorimetric assay using 96-well plates (Gunderson et al., 2021). Briefly, hepatopancreas tissue (100 mg) was added to 100 µl of ice cold 5% sulfosalicylic acid (SSA) and homogenized using a Bullet Blender (Next Advance, Inc., BBUC3137) (4 min @ speed 7 @ 4°C, 10–20 zirconium oxide beads). Samples were then centrifuged (16,813g for 2 min @ 4°C) using an Eppendorf microfuge (Eppendorf 5418) and kept on ice until being assayed for GSH content. NADPH (280 mM), DTNB (10 mM), and GSH Reductase (from Baker’s yeast, diluted to 50 units/mL) were prepared in stock buffer (143 mM sodium phosphate, 6.33 mM Na2EDTA (pH 7.5)). Supernatant from the hepatopancreas homogenate (2.5 µl) was added to a well containing 100 µl of NADPH, 15 µl of GSH reductase, and 32.5 µl of dH₂0 and allowed to incubate at 30°C for 10 min. DTNB (15 µl) was then added to each well and the absorbance read @ 412 nm (BioRad Benchmark Plus © spectrophotometer microplate reader) after a 10 min incubation at room temperature. GSH content (nmol GSH/gram tissue) was quantified using a GSH standard curve (0–19.5 µM).

2.3. Metallothionein (MT)

A DTNB (5,5’-dithiobis(nitrobenzoic acid)) based colorimetric assay was used to measure metallothionein concentrations as previously described (Gunderson et al., 2021; Gunderson et al., 2018; Gunderson et al., 2016; Linde and Garcia-Vazquez, 2006).

2.3.1. Sample Preparation

Hepatopancreas tissue (0.3 grams) was homogenized in 1 ml of homogenization buffer (0.5 M sucrose, 20 mM Tris HCl, 0.01% β-mercaptoethanol, pH 8.6) using a Bullet Blender (Next Advance, Inc., BBUC3137) (4 min @ speed 7 @ 4°C, 10–20 zirconium oxide beads). The homogenate was then centrifuged at 39,086 g (4 °C) for 20 min (L8–80M Beckman Ultracentrifuge). Cold absolute ethanol (1.05 ml, −20°C) and chloroform (80 µl) were then added to the resulting supernatant (1 ml). Samples were centrifuged at 3,716 g for 20 min (4°C) (Sorvall Legend RT centrifuge). Cold ethanol (3:1 v/v) was added to the resulting supernatant and the samples stored at −20°C for at least 1 hour. The samples were then centrifuged (3,716 g for 20 min) and the resulting pellet washed with 100 µl of an ethanol:chloroform:homogenization buffer mix (87:1:12). Samples were again centrifuged (3,716 g for 20 min), and the resulting pellets dried under a steady stream of nitrogen gas. The dried pellets were then resuspended in 300 µl of resuspension buffer (5 mM Tris-HCl, 1 mM EDTA, pH 7). The resuspended fraction was then added to 4.2 ml of DTNB (0.43 mM in 0.2 M sodium phosphate buffer, pH 8), vortexed thoroughly, and incubated for 30 min at room temperature. The absorbance of the samples was then measured in duplicate (sample divided in half) at 412 nm in a 24-well plate (BioRad Benchmark Plus © spectrophotometer microplate reader). MT concentrations (nmol MT/gram tissue) were quantified as previously described using a GSH reference curve (0–0.3 mM, 1 cysteine/GSH molecule), with the assumption that crayfish have 18 cysteine residues per MT molecule (Faria et al., 2010; Linde and Garcia-Vazquez, 2006).

2.4. Glutathione S-transferase Activity (GST)

Total GST activity (EC 2.5.1.18) was quantified using a previously published kinetic colorimetric 96-well plate assay that measures the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) to GSH (Gunderson et al., 2021; Gunderson et al., 2018; Gunderson et al., 2004).

2.4.1. Sample Preparation

Hepatopancreas tissue (0.33 g) was homogenized in 1 ml of homogenization buffer (100 mM Tris (pH 7.4), 0.1 M KCl, 1 mM EDTA, 1 mM PMSF, 5 µM aprotinin, 10 µM pepstatin) using a Bullet Blender (Next Advance, Inc., BBUC3137) (4 min @ speed 7 @ 4°C, 10–20 zirconium oxide beads). The cytosolic enzymes were isolated by a series of centrifugations. The homogenate was first centrifuged at 9,491 g (15 min, 4°C) using an Eppendorf microfuge (Eppendorf 5418). The resulting supernatant was then transferred to a second tube and centrifuged a second time at 14,326 g (20 min, 4°C). The supernatant from the second spin was then centrifuged at 100,000 g (60 min, 4°C) using a Beckman L8–80 M Ultracentrifuge. The supernatant from the final spin was aliquoted and stored at −80°C until assayed for GST activity.

2.4.2. GST Activity Assay

The total protein concentration for each cytosol sample was determined using a Bradford assay (Bradford, 1976). Cytosol samples (2.5 µg; total sample volume 20 µl) were added to a well containing 190 µl of 0.1 M phosphate buffer (pH 6.5) and 20 µL 12.5 mM GSH and allowed to incubate for 10 min at room temperature. CDNB was then added to the well and the absorbance was read immediately using a kinetic protocol (20 readings @ 11 sec intervals with a 1 sec mix time @ 340 nm) on a microplate reader (BioRad Benchmark Plus ©). GST activity was calculated using Beer’s law (A = Ɛlc) with an extinction coefficient of 9600/cm and reported as nmol GSH conjugated/(min*mg total protein) as previously described (Gunderson et al., 2004).

2.5. Statistical analyses.

Datasets were determined to be parametric, or non-parametric, using Shapiro-Wilk and Brown-Forsythe (or a two-sample test for variance) tests for normality and homogeneity of variance, respectively. Sex-differences were not observed in control animals for any of the endpoints examined (unpaired t-test, p > 0.05), so males and females were grouped for all analyses. Two-Way ANOVA (GST, parametric) or Kruskal-Wallace ANOVA (GSH and MT, non-parametric) were used to examine whether differences existed among treatment groups for each endpoint. An unpaired t-test was used to further examine whether a difference between the saline control and dimethoate treatment groups existed for MT concentrations, GSH concentrations, and GST activity, since a previous study by our research group reported inhibition of GST by dimethoate in crayfish collected from a different population and other studies report modulations of other endogenous antioxidants (that included GSH) by dimethoate (Ben Amara et al., 2011a; Ben Amara et al., 2011b; Gunderson et al., 2018; Sharma et al., 2005b). OriginPro 2020b (OriginLab Corporation) was used for all analyses.

3. Results

None of the treatments modulated GSH concentrations (KW ANOVA, p = 0.26) or MT concentrations (KW ANOVA, p = 0.89) in hepatopancreas tissue at the concentrations tested in this study (Figures 1 & 2). Furthermore, selenium (p = 0.19) and dimethoate (p = 0.4) were not significant factors influencing GST activity, and no interaction between the treatments was observed (p = 0.41), as determined using a Two-Way ANOVA (Figure 3). No significant differences between saline and dimethoate treatments existed in GSH concentrations (t-test, p = 0.42), MT concentrations (t-test, p = 0.37), or GST activity (t-test, p = 0.06), although a pattern suggesting decreased GST activity was observed (Figures 1,2, & 3).

Figure 1:

GSH concentrations (mean +/−S.D.) in hepatopancreas tissue collected from signal crayfish treated with saline vehicle (control) or selenium (Se: 0.1, 0.5, & 1.0 µg g⁻¹) with (+D) or without (−D) 0.9 µg g⁻¹ dimethoate. No significant differences (KW ANOVA, p > 0.05) were observed among the treatment groups.

Figure 2:

MT concentrations (mean +/− S.D.) in hepatopancreas tissue collected from signal crayfish treated with saline vehicle (control) or selenium (Se: 0.1, 0.5, & 1.0 µg g⁻¹) with (+D) or without (−D) 0.9 µg g⁻¹ dimethoate. No significant differences (KW ANOVA, p > 0.05) were observed among the treatment groups.

Figure 3:

GST activities (mean +/− S.D.) in hepatopancreas tissue collected from signal crayfish treated with saline vehicle (control) or selenium (Se: 0.1, 0.5, & 1.0 µg g⁻¹) with (+D) or without (−D) 0.9 µg g⁻¹ dimethoate. Selenium and dimethoate were not significant factors influencing GST activity, and no interaction between the treatments were observed (Two-Way ANOVA, p > 0.05).

4. Discussion

Endogenous antioxidant systems play an important role in an organism’s ability to manage and adapt to oxidative stress. In this study, we investigated whether selenium modulates the detoxification and endogenous antioxidant pathways associated with GSH, GST, and MT as well as examined if selenium interacted with dimethoate in the regulation of these endpoints. We hypothesized that selenium would modulate these endpoints and, given the fact that selenium can increase endogenous antioxidant capacity, ameliorate the inhibition of GST by dimethoate, an effect previous reported by our research group (Gunderson et al., 2018). Our predictions were not supported by the results from this study. First, selenium did not modulate GSH, GST activity, or MT concentrations at the concentrations tested. Secondly, dimethoate did not alter GSH and MT concentrations or lead to a significant decrease in GST activity as previously reported in a different crayfish population on the Boise River (Idaho, USA) (Gunderson et al., 2018). Finally, dimethoate and selenium cotreatments did not significantly (p > 0.05) modulate GSH, GST, or MT, although a pattern suggestive of selenium having a protective effect on dimethoate induced inhibition of GST activity was apparent.

4.1. GSH

GSH is the most abundant non-thiol protein in the cell and can be enzymatically attached to xenobiotics in conjunction with GST or act nonenzymatically to react with electrophilic compounds, thus acting as an antioxidant (Ulrich and Jakob, 2019). GSH is detectable in crayfish tissues, can be modulated (increased or decreased) by ROS generating compounds, and is often used as a biomarker indicative of exposure to pro-oxidants or when assessing the glutathione redox status of an organism (Gunderson et al., 2021; Gunderson et al., 2018; Kovacevic et al., 2008; Ulrich and Jakob, 2019). Selenium has been reported to both modulate GSH concentrations as well as strengthen antioxidant systems, thus providing a protective effect against oxidative stress. GSH activity increased in sea bream (Pagrus major) exposed to selenium (Kim and Kang, 2015). Another study demonstrated that selenium treatment increased GSH (4-fold) in blue mussels (Mytilus edulis), a response that contributed to the upregulation of antioxidant systems and subsequent protection against oxidative damage caused by copper sulfate exposure (Trevisan et al., 2011). Another study conducted in fish (Brycon cephalus) demonstrated that methyl parathion (organophosphate pesticide) exposure resulted in increases in SOD, CAT, GST, and LPO and decreases in GSH-Px and GSH, all of which are indicative of increased oxidative stress. Selenium supplementation reversed the adverse effects with no change in LPO being observed and GSH concentrations being maintained (Monteiro et al., 2009). Studies in Wistar rats have demonstrated that dimethoate exposure leads to decreases in GSH, Na⁺-K⁺-ATPase, AChE, and butyrylcholinesterase (cerebral cortex tissues), increases in GSH-Px and SOD (cerebral cortex tissues), increases in malondialdehyde (MDA) and lactate dehydrogenase (LDH) (liver tissue), and decreases in GSH, GSH-Px, SOD, and CAT (liver tissue) (Ben Amara et al., 2011a; Ben Amara et al., 2011b). Interestingly, selenium and/or vitamin E diminished the adverse effects presumably through their role as antioxidants. Previous work by our research group demonstrated that GSH was not modulated by mercury, zinc, copper, or dimethoate at the concentrations tested (Gunderson et al., 2021; Gunderson et al., 2018). In this study, GSH concentrations did not change in response to any of the treatments. All three of these studies suggest that the concentrations tested were either not high enough to elicit sufficient ROS production or that the endogenous antioxidant systems in these animals were robust enough to scavenge free radicals that were generated.

4.2. MT

MTs are non-enzymatic cysteine rich proteins that play numerous cellular roles that include sequestration and excretions of essential (i.e. Zn, Cu) and nonessential metals (Hg, Cd, Ag), serving as a reservoir for essential metals (i.e. Zn, Cu), maintaining metal homeostasis, and contributing to the overall antioxidant status of an organism (Amiard et al., 2006; Kouba et al., 2010; Newman, 1998). MTs are detectable in numerous taxa (including crayfish) and induced by metals, making them common markers to assess metal exposure in ecotoxicology studies (Amiard et al., 2006). Selenium induced MT in rats, although the mode of exposure influenced the response with injection leading to significant increases in hepatic and renal MT whereas dietary exposure did not lead to changes in hepatic MT (Chen and Whanger, 1994). MT increased in both gill and liver in tilapia (Oreochromis mozambicus) exposed to selenium, indicating that it is a suitable marker for selenium exposure in ecotoxicology studies in this species (Gobi et al., 2018). MT concentrations increased in response to mercury, zinc, and copper with tissue specific patterns being evident in signal crayfish (Pacifastacus leniusculus) (Gunderson et al., 2021; Gunderson et al., 2018). Dimethoate exposure (0.3,0.6,0.9 µg/kg) had no effect on MT concentrations in this species (Gunderson et al., 2018). MT did not change in response to selenium, dimethoate, or selenium-dimethoate treatments in this study. As noted above, the concentrations tested are either not high enough to modulate MT or the pathways associated with managing metals, pesticides, and oxidative stress were healthy enough to manage the exposure to these metals and compounds.

4.3. GST

GSTs are phase II enzymes that conjugate GSH to xenobiotics thus increasing the polarity of the molecules and facilitating clearance from the body via urine (Newman, 1998; Park et al., 2020). It is proposed the GSTs evolved as adaptations enabling organisms to manage stress due to xenobiotic and pro-oxidant exposure (Park et al., 2020). The NRF2 transcription factor (nuclear factor erythroid 2-related factor 2), considered to be a master controller of stress response genes, regulates GST expression along with antioxidant or electrophilic response elements (ARE/EpRE) in some cases (Park et al., 2020). Red swamp crayfish (Procambarus clarkii) fed diets supplemented with selenium demonstrated changes in antioxidants (Dorr et al., 2008). Furthermore, GST activity increased in sea bream (Pagrus major) exposed to selenium (Kim and Kang, 2015). Selenium exposure resulted in increased GST activity in both the liver and gill tissue of tilapia (Oreochromis mozambicus) (Gobi et al., 2018). In this study, GST activity was not modulated by selenium at the concentrations tested.

At the outset of the study, we predicted that dimethoate would decrease GST activity based on previous work by our research group, and that selenium cotreated with dimethoate would diminish this effect (Gunderson et al., 2018). While dimethoate did not decrease GST activity as predicted, a pattern of decreased activity is suggested (p = 0.06) and should not be ignored. Furthermore, GST activity did not differ from controls in crayfish co-exposed to selenium-dimethoate. These results warrant further investigation as a pattern exists, although not significant at p < 0.05, that supports our hypothesis.

The antioxidant capacity of populations can be influenced by numerous biotic and abiotic factors that are undoubtedly unique to a given population and could provide a potential explanation for the results reported in this study (Acs et al., 2016; Elia et al., 2006; Fisker et al., 2016; Kerambrun et al., 2016; Louiz et al., 2017). Previous work by our research group demonstrated that dimethoate (0.3,0.6, and 0.9 µg kg⁻¹) decreased GST activity in signal crayfish collected from a different population (Boise River, Idaho USA) in June. As noted above, we did not observe a decrease in GST activity (p = 0.06) in animals exposed to a higher dose of dimethoate (0.9 µg g⁻¹) in this study. Animals were collected from a localized region of the Snake River (Idaho, USA) in October, although they were acclimated to a summer light cycle for at least two weeks in the laboratory before being treated. The Snake River flows through areas impacted by anthropogenic activities (agriculture, etc.) that could increase the exposure of crayfish to pro-oxidants, thus increasing endogenous antioxidant defense mechanisms through adaptive responses. Ultimately, this could enable them to mitigate the adverse effects of oxidative stress, thus requiring even higher doses of dimethoate than that tested in this study to elicit an effect in animals from this population. Furthermore, seasonal variation in an organism’s ability to cope with pro-oxidant exposure could provide another explanation for the differences observed in the response of GST activity to dimethoate exposure reported in this study, given the fact that endogenous antioxidant expression is known to vary throughout the year in crayfish, crabs, and amphipods (Barim-Oz et al., 2017; Elia et al., 2006; Paital and Chainy, 2013; Sroda and Cossu-Leguille, 2011). Future studies designed to examine the effects of season and the response of different populations of crayfish to chemical stressors would add interesting information to the scientific literature reporting on the regulation of endogenous antioxidants by environmental contaminants

4.4. Conclusions

In this study, none of the endpoints examined changed in response to selenium, dimethoate, or selenium-dimethoate co-treatments. GSH, GST, and MT change in response to pro-oxidants and are thus commonly used as biomarkers indicative of contaminant exposure in ecotoxicology studies (Amiard et al., 2006; Dickinson and Forman, 2002; Park et al., 2020). Variations among taxa exist in the responses of these markers to metals, xenobiotics, and pro-oxidants thus necessitating the careful characterization of their regulation in a given model organism when applying them to field studies (Amiard et al., 2006; Dobritzsch et al., 2020). Some of the reported variability in responses within and among species may in part be explained by population differences in antioxidant capacity, detoxification enzyme expression, and seasonal variation. Finally, while our results did not support our initial hypotheses, they are suggestive and further investigation of the protective role of selenium in pro-oxidant exposure in this species is warranted given the limited scope of the experiment. Future studies that consider a broader range of time points, higher treatment doses of dimethoate and selenium, and examination of additional endpoints (i.e., individual GST isoforms, selenoproteins, and mRNA levels of NRF-2 targets) would all be interesting and potentially provide a different perspective on the results reported in this study. Population-specific responses as well as seasonal variations in endogenous antioxidant expression should also be considered in future experiments.

Highlights:

• Selenium did not modulate GSH, MT, or GST.

• Dimethoate exposure had no effect on GSH or MT concentrations.

• Dimethoate did not decrease GST in this population, although a suggestive pattern existed (p = 0.06).

• Selenium-dimethoate cotreatment did not alter GSH, MT, or GST, although a protective effect of selenium is suggested.