Impact of ethion and silver nanoparticles on oxidative stress markers in developing avian embryos: A dose-dependent analysis

Introduction

Pesticides play a critical role in modern agriculture, aiding in the control of pests, weeds, and diseases, thereby enhancing crop yields and ensuring food security (Anani et al., 2020) [1] . Over the past decades, the global use of pesticides has surged, with recent statistics indicating that more than 2 million tons are applied worldwide annually (Sharma et al., 2019) [2] . Among the different classes, organophosphates (OPs) remain one of the most frequently used, accounting for about 40% of all pesticides applied, mainly due to their effectiveness, relatively low persistence in the environment, and reduced risk of bioaccumulation compared to older pesticide classes like organochlorines (Goeb et al., 2020) [3] . Ethion, a widely used organophosphate insecticide, is primarily employed to protect crops such as citrus fruits, cotton, and vegetables from a variety of pests (Ahmad, 2008) [4] . Although effective, ethion poses significant risks to non-target organisms, including birds, fish, and mammals (Roney et al., 2000) [5] . The toxicological effects of ethion are primarily attributed to its ability to inhibit acetylcholinesterase activity, leading to the accumulation of acetylcholine at nerve synapses (Dewan et al., 2008) [6] . This, in turn, affects the nervous system and can lead to various health issues. Importantly, ethion has also been shown to induce oxidative stress by generating reactive oxygen species (ROS) that cause damage to cellular components and disrupt the balance of antioxidant defense mechanisms, leading to alterations in crucial antioxidant enzymes such as catalase, superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx) (Singh et al., 2006) [7] . Understanding these effects is vital for assessing the potential impact of ethion on the health of exposed organisms (Foster et al., 2004) [8] . Silver nanoparticles (AgNPs) have emerged as a versatile nanomaterial with a wide range of applications due to their excellent antimicrobial, antifungal, and antiviral properties (Galdiero et al., 2011) [9] . In agriculture, AgNPs are increasingly used to combat plant pathogens, reduce crop diseases, and improve growth, acting as nano-fertilizers or protective agents (Anand and Bhagat, 2019) [10] . Their use in animal husbandry has also been explored, where they help improve livestock health and prevent infections, often serving as a potential alternative to conventional antibiotics (Franci et al., 2015) [11] . Additionally, they have shown promise in enhancing feed efficiency and supporting overall animal productivity (Saleh and El-Magd, 2018) [12] . Despite these advantages, excessive exposure to silver nanoparticles can pose health risks. At high concentrations, AgNPs can disrupt cellular functions, induce oxidative stress, and impair antioxidant defense systems, leading to cellular toxicity (Kim and Ryu, 2013) [13] . This toxicity is of particular concern because it may damage vital organs, such as the liver and kidneys, by generating reactive oxygen species (ROS) and affecting overall redox balance (Wang et al., 2017) [14] . Hence, understanding the dose-dependent effects of silver nanoparticles is crucial when considering their application, especially in combination with pesticides like ethion (Tiwari et al., 2011) [15] . Therefore, the present study was carried out to investigate the effects of ethion and silver nanoparticles on oxidative stress markers in developing avian embryos (Patlolla et al., 2015; Bhatti et al., 2011) [16,17] . The aim was to evaluate how exposure to ethion, either alone or in combination with silver nanoparticles, impacts key oxidative stress biomarkers such as lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GPx) (Wen et al., 2022) [18] . Understanding these effects is essential, as oxidative stress plays a significant role in cellular damage, leading to potential toxicity and developmental abnormalities in avian embryos (Rezaei et al., 2018) [19] . By assessing the changes in these oxidative stress markers, this study sought to elucidate the extent to which ethion induces oxidative damage and how silver nanoparticles, at varying doses, either ameliorate or exacerbate this damage (Reshi et al., 2017; Elfaky et al., 2022) [20,21] . The findings from this research will provide valuable insights into the potential protective or toxic effects of silver nanoparticles against pesticide-induced oxidative stress, contributing to a better understanding of their combined impact on avian development (Nasir et al., 2015) [

Materials and Methods

Chemicals and Reagents

Ethion was obtained from Cheminova Industries Pvt. Ltd. (Mumbai, India), while all other chemicals were of analytical grade and sourced from reputable suppliers. Silver nanoparticles (AgNPs) were synthesized following the method described by (Yerragopu et al., 2020) [23] .

Egg Procurement and Preliminary Preparation

Fertile White Leghorn eggs were acquired from the Instructional Livestock Farm Complex (ILFC) at the College of Veterinary and Animal Sciences, Pookode, Wayanad, Kerala, following ethical approval (IAEC/COVAS/PKD/21/01/2023 dated 12.07.2023). The eggs were cleaned with 70% alcohol, allowed to air dry, weighed to approximately 50±5 grams, and appropriately labelled. Defective eggs were identified and removed by candling, and the air cell position was marked. The eggs were then incubated at 37.8°C with 55% humidity and turned every four hours (Adame and Ameha, 2023) [24] .

Yolk Sac Injection Method

On the fifth day of incubation, 120 eggs were candled to clearly identify the vitelline vessels. Each egg was injected with a 100 µL solution containing ethion, silver nanoparticles, or their combinations directly into the yolk sac (Bhanja et al., 2004) [25] . The eggs were distributed into ten distinct groups: a control group, groups treated with silver nanoparticles, groups treated with ethion, and groups receiving their combinations. Details of the experimental grouping are provided in the table below.

Experimental Design and Group Allocation

The study involved 10 groups of 12 embryonated eggs, to investigate the effects of silver nanoparticles and ethion, individually and in combination, on avian embryo development. Group 1 served as the control and received 100 µL of olive oil. Groups 2, 3, and 4 were treated with silver nanoparticles at doses of 25, 50, and 100 µg/egg, respectively, while Groups 5, 6, and 7 received ethion at doses of 73.5, 147, and 294 µg/egg in 100 µL olive oil. The acute toxicity of ethion in avian embryos was assessed on the fifth day of incubation using the method outlined by Randhawa (2009) [26] . Fifty embryos were divided into five groups, each containing ten embryos, and five different doses of ethion, ranging from 0.5 to 3.5 mg/egg, were administered via the yolk sac route. Each dose was delivered in a constant volume of 100 µL per egg. The embryos were observed for mortality at 30 minutes, 2 hours, 6 hours, 24 hours, and up to 14 days post-inoculation. Mortality was recorded after every observation, and the percentage of mortality was calculated to determine the LD50 values. Groups 8, 9, and 10 were administered combined treatments: AgNP-25 µg/egg + ETH-73.5 µg/egg, AgNP-50 µg/egg + ETH-147 µg/egg, and AgNP-100 µg/egg + ETH-294 µg/egg, respectively. Injections were given on the 5th day of incubation, and the eggs remained incubated until day 19, when they were opened for analysis of oxidative stress biomarker, as detailed in Table 1.

Assessment of Oxidative Stress Biomarkers

On the 19th day of incubation, liver samples were collected for analysis of oxidative stress biomarkers. A 10% liver homogenate was prepared in RIPA buffer, centrifuged at 3000 rpm for 15 minutes, and the supernatant was used to analyze oxidative stress markers, including lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), glutathione reduced (GSH), and glutathione peroxidase (GPx). (Guo et al., 2022) [27] .

Statistical Evaluation

The data were presented as Mean ± SEM (n=12). Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, utilizing SPSS Version 24.0 software.

Results and Discussion

The measurement of oxidative stress parameters is crucial in experimental studies as it provides valuable insights into the cellular and biochemical effects of various substances, including toxins and therapeutics (Milatovic et al., 2006) [28] . Oxidative stress arises from an imbalance between reactive oxygen species (ROS) production and antioxidant defenses, leading to potential damage to lipids, proteins and DNA (Loft and Poulsen, 1999) [29] . By assessing markers such as lipid peroxidation, superoxide dismutase, catalase and glutathione levels, researchers can evaluate the extent of oxidative damage and the effectiveness of antioxidant responses in biological systems (Enkvetchakul et al., 1993) [30] . This is particularly significant in studies of environmental pollutants like pesticides and nanomaterials, as it helps elucidate their mechanisms of toxicity and the potential for protective strategies (Manke et al., 2013) [31] . Understanding oxidative stress dynamics allows for a more comprehensive assessment of health risks and notifies the development of mitigation approaches in both agricultural and medical contexts (Costantini, 2019) [32] . In addition to its acetylcholinesterase (AChE) inhibitory activity, oxidative stress induction plays a pivotal role in the mechanism of action of ethion (Durom et al., 2024) [33] . Ethion, an organophosphate pesticide, exerts its toxic effects not only by disrupting neurotransmission but also by generating reactive oxygen species (ROS) that overwhelm the cellular antioxidant defense system (Shukla et al., 2021) [34] . This oxidative stress can lead to lipid peroxidation, protein oxidation, and DNA damage, contributing to cellular dysfunction and apoptosis (Bhatti et al., 2011) [17] . Elevation of ROS levels are linked to various pathological conditions, including neurodegenerative diseases and developmental disorders in embryos (Micangeli et al., 2022) [36] . Consequently, the oxidative stress induced by ethion may exacerbate its neurotoxic effects, leading to more severe developmental consequences in avian embryos (Ranjith et al., 2024) [38] . Understanding this dual mechanism enhances our comprehension of ethion's toxicity and underscores the need to evaluate both its enzymatic inhibition and oxidative stress contributions in toxicological assessments (Cortés-Iza and Rodríguez, 2018) [37] . Ethion administration significantly increases lipid peroxidation (Table 2), as indicated by the elevated levels of malondialdehyde (MDA), a key byproduct of lipid oxidation (Bhatti et al., 2010) [39] . This occurs primarily through the generation of reactive oxygen species (ROS) that result from ethion's toxic effects (Tuncmen and Tüzmen, 2007) [40] . When ethion is metabolized, it can lead to an imbalance between the production of ROS and the antioxidant defenses, resulting in oxidative stress (Guendouz et al., 2023) [42] . This oxidative stress initiates the peroxidation of polyunsaturated fatty acids in cell membranes, leading to increased MDA formation (Bhatti et al., 2011) [17] . At low and moderate doses of ethion, the co-administration of silver nanoparticles (AgNPs) appears to have a protective effect, likely due to the nanoparticles' potential antioxidant properties (Reshi et al., 2017) [20] . Silver nanoparticles can scavenge ROS, reducing oxidative stress and thus limiting lipid peroxidation (Reshi et al., 2020) [44] . This suggests that at these lower doses, AgNPs may enhance the embryo's antioxidant defenses, leading to no significant change or even amelioration of ethion's toxic effects (Sreelekha et al., 2021) [45] . However, at high doses of AgNPs, the situation changes. High concentrations of AgNPs may exert prooxidant effects, overwhelming the antioxidant capacity and contributing to further oxidative stress (Kim and Ryu, 2013) [13] . This could result from increased synthesis of ROS or from the saturation of the antioxidant systems, leading to increased lipid peroxidation and higher levels of MDA (Khan et al., 2017) [47] . The combination of high-dose AgNPs with high-dose ethion exacerbates the oxidative stress, resulting in an additive or synergistic toxic effect that which manifests as increased lipid peroxidation (Özkul et al., 2021) [48] . Reduced Glutathione (GSH) in the present study showed a dose-dependent reduction in ethion and AgNP-treated groups when compared to control groups (Table 2). Reduced Glutathione (GSH) is a tripeptide composed of glutamine, cysteine and glycine, and it plays a pivotal role as a major antioxidant in cells (Hussain et al., 1999) [56] . GSH protects cells by neutralizing reactive oxygen species (ROS) and maintaining the redox state of the cell. It acts as a substrate for various enzymes and is crucial for detoxification processes (Yuan and Kaplowitz, 2009) [57] . Superoxide Dismutase (SOD) catalyzes the dismutation of superoxide radicals (O₂⁻), converting them into hydrogen peroxide (H₂O₂) and molecular oxygen (O₂). By doing so, SOD helps to mitigate the harmful effects of superoxide, which can cause cellular damage if allowed to accumulate (Arora et al., 2008) [51] . Catalase further processes hydrogen peroxide produced by SOD, converting it into water (H₂O) and oxygen (O₂). This enzyme is essential for preventing the accumulation of H₂O₂, which can also lead to oxidative damage if levels are not adequately controlled (Docea et al., 2020) [52] . In the ethion-treated group, the observed decrease in both catalase and SOD levels indicates that ethion induces oxidative stress, overwhelming the antioxidant defense system (Bhatti et al., 2011). Ethion may promote the production of reactive oxygen species (ROS), which can damage cellular components and hinder the normal expression or activity of these protective enzymes (Singh et al., 2006) [7] . In the groups co-administered with low and moderate doses of silver nanoparticles alongside ethion, the normalization of catalase and SOD levels suggests that the silver nanoparticles enhance the antioxidant capacity of the developing embryos (Reshi et al., 2020) [44] . By reducing oxidative stress, AgNPs help restore the activity of these enzymes, allowing them to function effectively in mitigating further oxidative damage (Sreelekha et al., 2021) [45] . However, in the groups receiving high doses of silver nanoparticles, both catalase and SOD levels decrease again (Shayesteh et al., 2014) [53] . This could indicate that excessive silver nanoparticles may induce pro-oxidative effects, leading to further ROS production that overwhelms the antioxidant defences (Gan et al., 2020) [54] . When combined with high doses of ethion, the resulting oxidative stress may be too severe for the antioxidant systems to cope with resulting in decreased enzyme levels (Abdel-Gawad et al., 2021) [55] . This highlights the importance of dose management in therapeutic applications, as both low and moderate doses can be protective, while high doses can be detrimental (Reshi et al., 2017) [20] . Glutathione Peroxidase (GPx) is an enzyme that catalyzes the reduction of hydrogen peroxide (H₂O₂) and organic hydroperoxides, using GSH as a reducing agent (Ben Amara et al., 2015) [58] . This reaction converts H₂O₂ into water, thereby protecting the cell from oxidative damage (Little et al., 1970) [59] . In the ethion-treated group, the decrease in both GSH and GPx levels indicates that ethion exposure induces oxidative stress, leading to glutathione depletion and reduced GPx activity (Bhatti et al., 2010) [39] . This suggests that ethion may either enhance the production of ROS or interfere with the synthesis and recycling of GSH, compromising the antioxidant defense (Singh et al.,2006) [7] . In groups receiving low and moderate doses of (AgNPs) alongside ethion, the restoration of GSH and GPx levels implies that AgNPs have a protective effect against oxidative stress (Reshi et al., 2020) [44] . They may enhance the synthesis or regeneration of GSH, thereby allowing GPx to function effectively and reduce hydrogen peroxide levels (Reshi et al., 2017) [20] . However, in the high-dose silver nanoparticles groups, both GSH and GPx levels decline again (Ranjbar et al., 2014) [60] . This could suggest that at elevated doses, AgNPs may exert a pro-oxidative effect, leading to increased ROS that depletes GSH and overwhelms GPx (Yin et al., 2013) [61] . When combined with high doses of ethion, cumulative oxidative stress may exceed the antioxidant system's capacity, resulting in decreased levels of these critical enzymes (Shukla et al., 2021) [34] . This highlights the need for careful dosage considerations, as both insufficient and excessive doses can impair the antioxidant response (Ranjith et al., 2022) [62] . Overall, the differing effects observed in low versus high doses of silver nanoparticles highlight the complex interplay between dose-dependent responses and the mechanisms of oxidative stress (Park et al., 2010) [49] . While lower doses may provide protective benefits, higher doses can exacerbate oxidative damage, underscoring the importance of dose considerations in toxicological studies (Williams et al., 2016) [50] . In summary, the study investigated the effects of ethion and silver nanoparticles (AgNPs) on oxidative stress markers in developing avian embryos. Ethion exposure significantly elevated lipid peroxidation, indicated by increased malondialdehyde levels, reflecting enhanced oxidative stress. While low to moderate doses of AgNPs coadministered with ethion normalized oxidative stress markers and mitigated ethion's toxic effects, high doses of AgNPs exacerbated lipid peroxidation and decreased antioxidant enzyme levels, including catalase, superoxide dismutase, glutathione, and glutathione peroxidase. These findings underscore the dual role of AgNPs as both protective agents at lower doses and potential stressors at higher concentrations, emphasizing the importance of dosage in managing oxidative stress in avian embryos.

Conclusion

In conclusion, this study highlights the detrimental impact of ethion on oxidative stress markers in developing avian embryos, demonstrating increased lipid peroxidation and reduced activity of key antioxidant enzymes. The use of silver nanoparticles revealed a complex, dose-dependent interaction: low to moderate doses were effective in ameliorating the toxic effects of ethion, whereas higher doses led to exacerbated oxidative stress. These findings suggest that while silver nanoparticles hold promise as protective agents against pesticide toxicity, their application must be approached with caution to avoid adverse effects. Further research is warranted to explore optimal dosing strategies and the mechanisms underlying these interactions, thereby contributing to safer agricultural practices and enhanced animal health.

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