Enzymatic and non-enzymatic detoxification in Lycosa terrestris and Pardosa birmanica exposed to single and binary mixture of copper and lead
Graphical abstract
Introduction
Copper (Cu) and lead (Pb) coexists in the soils of agroecosystem due to the application of pesticides, fertilizers, sewage sludge, animal manure and irrigation of crops with untreated water of industry and municipality (Waseem et al., 2014; Ali et al., 2015: Rizwan et al., 2016). Terrestrial invertebrates that live close to the contaminated areas can assimilate these metals into their bodies through different uptake routes like ingestion of food and soil, dermal absorption from soil and inhalation of contaminated air (Vaufleury and Pihan, 2002; Peijnenburg et al., 2012; Nica et al., 2013). Cu is an essential element for all organisms and serve as a key cofactor of many important enzymes, i.e., bis-mutase, pyruvate hydrolase, superoxide dismutase, prophenoloxidase, and cytochrome c oxidase, etc. (Valko et al., 2005; Huang et al., 2012; Bonnail et al., 2016). Nevertheless, excessive intake of Cu could induce oxidative stress and cause dysfunction and denaturation of enzymatic and structural proteins (Mirmonsef et al., 2017; Wilczek et al., 2018). Pb is a non-essential heavy metal for living organisms that exert toxic effects by producing reactive oxygen species (ROS) in cells and inducing oxidative damage. In addition, the accumulation of Pb inactivates many crucial enzymes, inhibits the absorption of other minerals and stops the synthesis of structural proteins (Patrick, 2006; Wu et al., 2016; Yin et al., 2019).
Organisms live in metal contaminated areas usually develop several defense mechanisms like sequestration, metabolization, detoxification, and excretion, which protect them from the toxicity of metals (Eraly et al., 2010). Terrestrial invertebrates have developed various enzymatic and non-enzymatic detoxification mechanisms to minimize the toxic effects of metals (Ečimović et al., 2018). The major contributors to the non-enzymatic detoxification process are reduced glutathione (GSH) and metallothionein (MTs) proteins. GSH is an important cytosolic antioxidant that neutralizes reactive oxygen species (ROS) through oxidation and protects cells from oxidative damage (Kovačević et al., 2008). It also takes part in the detoxification process as a cofactor of different glutathione-dependent enzymes such as GST and GPX (Perić-Mataruga et al., 2019). Metallothioneins (MTs) are ubiquitous cysteine-rich metals inducible non-enzymatic proteins (Amiard et al., 2006). These proteins perform detoxification through the regulation of homeostasis of biogenic metals (like, Zn, Cu) and by neutralizing non-essential metal (like, Cd, Ag, Hg) (Eraly et al., 2010; Babczyńska et al., 2011b).
The enzymatic defense system includes phase I and phase II biotransformation reactions. In phase I reactions different enzymes like cytochrome P450 (CYP 450), carboxylesterase (CarbE) and acetylcholinesterase (AchE) metabolize xenobiotic molecules (metals or pesticides) into less toxic form through hydrolytic, oxidative or reductive reactions (Yang et al., 2012; Wu et al., 2016; Wilczek, 2017). The cytochrome P450 (CYP 450) monooxygenases belong to a large family of hemeproteins catalyzing the conversion of lipophilic and hydrophobic compounds into less toxic hydrophilic molecules for clearance in cells via oxidation (Yang et al., 2012; Gunderson et al., 2018). Carboxylesterase (CarbE) is another important first phase cytoplasmic hydrolase enzyme with wide specificity and detoxification ability. It catalyzes the hydrolysis of esterified xenobiotics into corresponding carboxylic acid and alcohol (Wheelock and Nakagawa, 2010). CarbE activity is well-characterized for its role in the detoxification of many agrochemicals and metals in several invertebrate’s taxa that include beetles, grasshopper, aphids, and earthworms (Wilczek et al., 2003; Augustyniak et al., 2005; Gao et al., 2014; Ečimović et al., 2018). Acetylcholinesterase (AchE) is a neurotransmitter hydrolase that helps in the transmission of nerve impulses by hydrolytic metabolism of acetylcholine into choline and acetate (Kim and Lee, 2018). Initially, it was used as a biomarker of many neurotoxic compounds like organophosphate and carbamate. However, its activity can also be affected by other xenobiotics, such as heavy metals (Van Praet et al., 2014; Bonnail et al., 2016).
In phase II reactions, detoxification enzymes like glutathione S-transferase, glycosyltransferases, sulfotransferases, and glycosidases catalyze the conjugation of phase I intermediates with their polar groups to form water-soluble and less toxic conjugates which can easily be excreted from the body (Regoli and Giuliani, 2014; Berenbaum and Johnson, 2015). Glutathione S-transferase (GST) is an important enzyme of Phase II reactions that catalyzes the conjugation reaction between endogenous nucleophilic GSH and electrophilic phase I intermediates for detoxification. (Vidal-Liñán et al., 2016; Sillero-Ríos et al., 2018). It is widely used as a potential biomarker of several invertebrates’ groups exposed to the complex mixture of contaminants in the environment (Świergosz-Kowalewska et al., 2006; Abdel-Halim et al., 2013; Dedeke et al., 2018).
Spiders are ubiquitous predators of terrestrial environment and well recognized for their role in the regulation of insect pest populations in the terrestrial ecosystem (Marc et al., 1999; Wise, 2006). In ecotoxicological studies, they are often used as potential bioindicators due to their high tolerances and physiological adaptability towards metal pollution (Babczyńska et al., 2012; Wilczek, 2017). Spiders have developed various enzymatic and non-enzymatic defense mechanisms against metals, which allow them to survive in heavily contaminated areas (Wilczek et al., 2004, 2008, 2013). These defense mechanisms depend on the age, sex, species, physiological status, and hunting strategies of exposed spiders (Wilczek et al., 2008; Babczyńska et al., 2012). Wolf spiders (Araneae: Lycosidae) are one of the most common and abundant predators of terrestrial ecosystem around the world (Tahir et al., 2011; Niedobová et al., 2016; Hansson et al., 2019). They are active hunters and inhabit the upper layer of the soil, which may be contaminated with several pollutants, including heavy metals. Therefore, these spiders may be exposed to pollutants via their food or water in the soil.
The present study was a follow-up of our previous investigation in which different antioxidant enzymes activities in spiders were measured as biomarkers of oxidative stress induced by Cu and Pb (Aziz et al., 2020b). The current research aimed to measure the response of various enzymatic and non-enzymatic detoxification mechanisms to gain more in-depth knowledge of the defense system of spiders activated by metals. For this purpose, the two most dominant ground-dwelling spider’s species Lycosa terrestris (Butt et al., 2006) and Pardosa birmanica (Simon, 1884) were simultaneously exposed to Cu, Pb singly or in a binary mixture (Cu + Pb) via food and soil and the activities of CYP 450, CarbE, AchE, and GST (enzymatic) and GSH and MTs (non-enzymatic) proteins were measured in spiders after different days of metal exposure.
Section snippets
Test organisms
The detailed procedure for the rearing of spider species, was previously described in Aziz et al. (2020a and 2020b). For the experiment, L. terrestris and P. birmanica were cultured in the laboratory on an artificial diet consisting of an egg yolk, soya milk (100 ml) and dairy milk (100 ml) (Amalin et al., 2001). The spiders were housed separately in transparent plastic containers (75 mm length ×55 mm width ×60 mm height) filled with 50 g of artificial soil of pH 6.0 ± 0.5 and maintained at its
Metal concentrations
The total Cu and Pb concentrations in soil, food and spiders’ species are presented in Table 1. The results showed that the Cu and Pb contents in both spiders’ species increased with increasing time of exposure in all experimental groups. In combined metal treatments, the accumulation of Cu from food decreased approximately two times as compared to Cu-only treatment in both species (F = 7.39, d.f. = 2, P = 0.001 for L. terrestris; F = 18.27, d.f. = 2, P < 0.001 for P. birmanica). Nevertheless,
Discussion
The present study signifies the importance of different enzymatic and non-enzymatic responses in two ground spiders L. terrestris and P. birmanica. Spiders species were treated with Cu and Pb singly or their binary mixture (Cu + Pb) through soil and food to assess the suitability of CYP 450, GST, CarbE, AchE, GSH, and MTs as biomarkers of metal exposure in studied species. Intriguingly, both spider species are capable of accumulating a high quantity of Cu and Pb in their bodies, which depends
Conclusion
The present study indicates that the accumulation of Cu and Pb can produce complex biochemical changes in spiders, which are associated with the type of metal, its accumulation level, and exposure duration. In Lycosa terrestris and Pardosa birmanica, the increase in metal accumulation also increased the levels of all enzymatic and non-enzymatic parameters with some exceptions. Firstly, the high accumulation of Cu caused a significant decrease in CYP 450 activity. Secondly, the AchE activity was
CRediT authorship contribution statement
Nida Aziz: Data curation, Formal analysis, Writing - original draft. Abida Butt: Conceptualization, Funding acquisition, Writing - review & editing.
Declaration of Competing Interest
The authors reported no declarations of interest.
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