Determination of Oxidative Stress Responses Induced by Copper Oxide (Cuo) Nanoparticle in Gammarus Pulex

Determination of Oxidative Stress Responses Induced by Copper Oxide (Cuo) Nanoparticle in Gammarus Pulex | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Determination of Oxidative Stress Responses Induced by Copper Oxide (Cuo) Nanoparticle in Gammarus Pulex Ayse Nur Aydin, Osman SERDAR, Işıl Canan Çiçek Çimen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3850318/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract As Copper Oxide (CuO) has a wide range of uses in industry, it is thought to have a wide polluting effect on the environment and aquatic environment. Gammarus pulex was chosen as the model organism in this study, which was carried out with the aim of realizing the effect of CuO mixed into the water environment. In order to carry out the study, CuO was exposed to 0 (control), 10, 20, 40 ppm concentrations for 24, 96 and 120 (elimination) hours. Samples were taken from the experimental environment at the end of 24, 96 and 120 hours. The study was carried out in 3 replicates. The assessment of oxidative stress and antioxidant biomarkers was conducted using ELISA kits obtained from CAYMAN Chemical Company. The parameters analyzed included the activity of SOD and CAT, as well as the levels of TBARS and GSH. The biochemical studies were evaluated using the one-way ANOVA (Duncan 0.05) feature of the SPSS 24.0 package program. With the study data, increases in SOD activities and fluctuations in CAT activity were determined. Decreases in GSH levels and increases in TBARS levels occurred. ds: Gammarus pulex Copper Oxide Oxidative stress Biomarkers Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION The exponential growth of nanotechnology has significantly amplified environmental apprehensions regarding the potential ecological hazards associated with engineered nanoparticles (NPs) (Syberg and Hansen, 2016 ). Extensive nanoecotoxicological research is enhancing our comprehension of the ecotoxicity of nanoparticles (Kahru and Dubourguier, 2010 ; Kunzmann et al., 2011 ; Petersen et al., 2015 ). Metal oxide nanoparticles (MNPs) are extensively utilized engineering nanomaterials (Vance et al., 2015 ). Copper oxide nanoparticles (CuO-NPs) find use in several industries including semiconductor devices, catalysts, photovoltaic cells, wood preservation, textile fibers, biocides, coatings, thermoplastics, colorants, and ceramic additives for optical glass polishing (Ma et al., 2017 ). The overabundance of pollutants in aquatic environments leads to substantial environmental and health issues (McNeil and Fredberg, 2011 ). Pollutants can elevate the concentrations of metals in natural water, leading to significant harm to both freshwater and marine ecosystems (Muhammed et al., 2011; Yu et al., 2011 ; El Nemr, 2012; El Nemr et al., 2012 ). The detrimental impacts of heavy metals mostly stem from their capacity to spread and multiply in aquatic food chains, as well as their toxicity and tendency to accumulate in biological tissues (Matta et al., 1999 ; İslam and Tanaka, 2004; Yi et al., 2011 ). Copper has considerable toxicity towards aquatic creatures, inducing irreversible harm at concentrations slightly surpassing those necessary for their growth and reproduction (Baldwin et al., 2003 ). The accumulation of waste from nanoparticles (NPs) in aquatic species, including fish, bacteria, single-celled organisms, crustaceans, and algae, leads to cellular damage and toxicity (Shaw and Handy 2011 ). Nanoparticles (NPs) have been found to accumulate in the organs of aquatic species, and their introduction into water can potentially modify the physiological reactions of these organisms (Chupani et al., 2018 ). The presence of CuO-NPs in the aqueous environment can lead to hazardous effects on aquatic organisms. This is mostly caused by the release of copper ions and nanoforms, which the organisms are exposed to (Gomes et al., 2011 ). Moreover, both enzymatic and non-enzymatic parameters are commonly employed to forecast the impact of hazardous chemicals on aquatic organisms. The impact of dangerous chemicals on the activity of enzymes is a crucial biochemical factor regulated by stress (Jahanbakhshi and Hedayati 2013 ). Oxidative stress is a crucial mechanism for protecting against harmful reactive oxygen species (ROS) or healing oxidative damage (Dorval and Hontela 2003 ). Copper is a metal that can help protect against pollutants by contributing to antioxidative defense (Parvez and Raisuddin 2006 ). Copper can impact oxidative stress due to its potential to enhance the production of reactive oxygen species (ROS) through a Fenton-like reaction, which is a significant factor in lipid peroxidation (Prousek, 2007 ). While tiny quantities of copper are necessary for all species and play a crucial function in the regulation of superoxide dismutase, it can be harmful to aquatic animals by depleting glutathione levels (Speisky et al., 2009 ). Copper exhibits redox potential and generates an abundance of free radicals, leading to the buildup of O2 and H2O (Moyson et al., 2016; Aliko et al., 2015). Additionally, it serves as a catalyst for Cu, SOD, CAT, and GST enzymes. Typically, the cell's favorable antioxidant and equilibrium are upheld by a mixture of diminutive antioxidant molecules, such as SOD, GST, and CAT enzymes (Glass et al., 1985 ). Oxidative stress arises from the impact of heavy metals that disturb the normal functioning of antioxidant enzyme systems (Dimitrova et al., 1994 ). Reactive oxygen species that are harmful to the body react with cellular components, resulting in the oxidation of proteins and damage to DNA through oxidation. This process leads to the deactivation of enzymes, tissue damage, rupture of cell membranes, mutations, and ultimately, cell death (Vutukuru et al., 2006 ). Bioindicators not only provide information about the deterioration caused by the accumulation of various pollutants in ecosystems, but also help determine how long the problems have persisted through physical and chemical tests performed in that ecosystem (Aydın et al., 2023 ). Organisms belonging to the Gammaridae family are employed as bioindicators in aquatic habitats (Serdar et al., 2018 ). Gammarids are bioindicators that are often present in freshwater ecosystems and are regularly employed in ecotoxicological investigations. These creatures serve as a primary food source for fish and amphibians and are abundant in streams (Aydın et al., 2022 ). G. pulex has been found to be susceptible to a range of contaminants, as demonstrated by Serdar et al. ( 2018 ) and Tatar et al. ( 2018 ). G. pulex was selected as the test organism for this study due to its ecological significance and role in ecosystems. This study aims to analyze the superoxide dismutase (SOD) and catalase (CAT) activity, thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) levels, as well as the absorption amounts of copper oxide nanoparticles (CuO NP), in the bioindicator species G. pulex . These parameters are indicators of oxidative stress in the aquatic ecosystem. 2. MATERIAL METHOD Nanoparticles NP materials used in the study were obtained from the CuO commercial company (SkySpring). The chemical, which is in the analytical reagent class, was used without any purification or purification. The shape and size data declared by the manufacturer for NP were used in bioassay studies with reference to the shape and size data declared by the manufacturer. Organism Provision and Adaptation G. pulex individuals used in the study were collected from the side branches of Munzur Stream in Tunceli province with the help of a bottom scoop and brought to the Munzur University Faculty of Fisheries research laboratory with air supplementation. G. pulex individuals were placed in 40x20x20 cm aquariums and adapted to laboratory conditions for 4 weeks. Environments suitable for natural habitats were created for the adaptation of G. pulex to laboratory conditions. For this purpose, sediments taken from the natural environment of G. pulex were washed with pure water and placed in stock aquariums. Water brought from the natural environment of G. pulex was added to the aquariums. Stock aquariums were supplemented with oxygen using an air engine. A 12:12 dark:light photoperiod was applied in ambient lighting. The ambient temperature was fixed at 18 ͦC with a thermostatic air conditioner and the aquarium water temperature with a chiller. After the adaptation environment was prepared, G. pulex collected from Munzur Stream were placed in stock aquariums. G. pulex was allowed to adapt to laboratory conditions. 70% of the water in stock aquariums was renewed weekly. To feed G. pulex , shrub willow tree leaves were collected and left to rot. Acute Toxicity Tests and Trial Design Experiments have not been conducted to determine sublethal concentrations in order to determine the acute toxicity value, taking into account the harm of pollutants to nature and the environment. Sublethal concentration values were determined by reviewing the literature and appropriate concentration values. In all experimental stages of the research, 0.5 liters of non-chlorinated water taken from the natural environment of the creatures was used in 1-liter glass aquariums. 10 G. pulex were placed in these aquariums for each concentration. Group Control; There is no retained CuO concentration in water taken from the natural environment of organisms. Group C1; 10 ppm CuO concentration to (CuO), Group C2; 20 ppm CuO concentration to (CuO), Group C3; (CuO), 40 ppm CuO concentration, To determine the changes in oxidative stress and antioxidant biomarker parameters during elimination, samples taken after 96 hours were kept in spring water brought from the living areas for 24 hours, and then the changes in oxidative stress and antioxidant biomarker parameters during elimination were determined. Biochemical analyzes Tissue samples collected at 24 and 96 hours were utilized. The samples were weighed and then mixed with PBS buffer (phosphate-buffered saline) at a ratio of 1 part sample to 5 parts buffer by weight. The mixture was then homogenized using an ice homogenizer in order to assess antioxidant properties. The samples underwent centrifugation at a speed of 17,000 revolutions per minute for a duration of 15 minutes. The resulting liquid portion, known as the supernatant, was then preserved in a deep freezer at a temperature of -86°C until further measurements were conducted. The activities of superoxide dismutase (SOD) and catalase (CAT), as well as the levels of thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH), were measured using ELISA kits obtained from CAYMAN Chemical Company. Statistical analysis SPSS 24.0 package program one-way ANOVA (Duncan 0.05) was used to evaluate biochemical analyses. 3. RESULTS SOD Activity The Fig. 1 displays the temporal variations of Superoxide Dismutase (SOD) activities in G. pulex when exposed to various concentrations of CuO. Significant increases in superoxide dismutase (SOD) activities were observed in all concentration groups of CuO at 24 and 96 hours, compared to the control group. The increases were statistically significant, with a p-value of less than 0.05. Significant changes, with a statistical significance level of p < 0.05, were detected when comparing the application groups with elimination groups. CAT Aktivity The Fig. 2 displays the CAT activities in G. pulex that were subjected to varying concentrations of CuO across different time periods. All groups experienced statistically significant (p < 0.05) declines after 24 hours, when compared to the control group. The statistical analysis conducted after 96 hours found that the differences observed between the application groups and the control group were not significant (p > 0.05). Negligible changes (p > 0.05) were seen when comparing the deletion groups and application groups. GSH Level The levels of glutathione (GSH) in G. pulex subjected to various doses of CuO at different time intervals are presented in Fig. 3 . Significant decreases (p < 0.05) were detected in all amounts of CuO at 24 and 96 hours compared to the control group, according to statistical analysis. Significant changes, with a statistical significance level of p < 0.05, were detected when comparing the elimination groups with application groups. TBARS Level The Fig. 4 presents the TBARS levels in G. pulex that were treated to various doses of CuO at different time intervals. Significant increases (p < 0.05) were seen in all groups using CuO at 24 and 96 hours, compared to the control group. Significant changes, with a statistical significance level of p < 0.05, were detected when comparing the elimination groups with application groups. 4. DISCUSSION Aquatic creatures, including mammals, have also developed the ROS-scavenging antioxidant defense mechanism. However, any disruption in this system might negatively impact their survival, growth, and physiology (Filho, 1996 ; Pandey et al., 2003 ). The detrimental effects induced by reactive oxygen species (ROS) are counteracted by three prominent antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Superoxide dismutase (SOD) and catalase (CAT) are the main mechanisms that protect cells from oxidative damage (Fang and Zheng, 2002 ). Superoxide dismutase (SOD) transforms harmful superoxide radicals into hydrogen peroxide (Halliwell and Gutteridge, 2007), which is then broken down by catalase (CAT) into molecular oxygen and water (Chelikani et al., 2004 ). In our study, increases in SOD activity and fluctuations in CAT activity are similar to many studies in the literature. Naeemi et al. ( 2020 ), stated that there were increases in SOD and CAT activity with the effect of CuO NP in Cyprinus carpio . Aziz et al. ( 2022 ), stated that there were increases in SOD activity and decreases in CAT activity as a result of CuO exposure in Hypophthalmichthys nobilis . Abdel-Latif et al. ( 2021 ), examined the effects of CuO on Oreochromis niloticus and observed that there were increases in SOD activities. Fazelian et al. ( 2019 ), stated that CAT activity increased with the effect of CuO on Nannochloropsis oculata . Abdel-Khalek et al. ( 2015 ), observed increases in SOD and CAT activities due to the CuO effect of O.niloticus . Srikanth et al. ( 2016 ), stated that SOD and CAT activity increased with the effect of CuO in Chinook salmon . Aziz and Abdullah, ( 2023 ) stated that there were increases in CAT activities in Labeo rohita due to the effect of CuO. Pradhan et al. ( 2016 ) stated that there were increases in SOD activities in Allogamus ligonifer exposed to CuO. Wang et al. ( 2019 ), stated that exposure to Cu NPs increased SOD and CAT activities in Takifugu fasciatus . Soliman and Ragab ( 2018 ), stated that SOD activity was increased by CuO in Poecilia reticulata . Dawood et al. ( 2020 ) observed that CuO increased SOD and CAT activities in C.carpio . Glutathione (GSH) has a crucial function as an antioxidant, preserving the integrity of proteins and enzymes, and acting as a cofactor for the GST enzyme. Several studies have documented a reduction in the levels of glutathione (GSH) in aquatic species following their exposure to metal ions (Loro et al., 2012 ). The findings suggests that the decline in GSH level in G. pulex is contingent upon the concentration of metals. The findings of our investigation are consistent with the research conducted by Abdel-Khalek et al. ( 2015 ), which demonstrated a reduction in GSH levels as a result of the impact of CuO on O.niloticus . Mwaanga et al. ( 2014 ), stated that CuO and ZnO NPs caused a decrease in GSH levels in D.magna . Akbary et al. ( 2018 ), stated that CuO reduced the GSH level on mullet. Srikanth et al. ( 2016 ), reported that GSH levels decreased in C. salmon with the effect of CuO. Villarreal et al. ( 2014 ), stated that decreases occurred in GSH levels due to the effect of O.mossambicus CuO. Liu et al. ( 2014 ), found that GSH level decreased in D.magna with the effect of CuO. Noureen et al. ( 2018 ) reported that CuO reduced GSH levels in C. carpio . Wang et al. ( 2015 ) stated that CuO caused decreases in GSH levels in Epinephelus coioides . Ogunsuyi et al. ( 2019 ), stated that GSH levels decreased with the effect of CuO on Clarias gariepinus . Soliman and Ragab ( 2018 ), stated that GSH level was reduced by CuO in Poecilia reticulata . Oxidative stress can lead to the degradation of membranes by causing peroxidation of lipid tails in the membrane structure. This process results in the formation of reactive chemicals such malondialdehyde (MDA) (Barata et al., 2005 ). ROS ultimately leads to lipid peroxidation. Lipid peroxidation results in the formation of organic hydroperoxides, which can be broken down into different organic compounds, such as MDA (Barata et al., 2005 ). This frequently results in a cyclic detrimental process, since certain byproducts of the breakdown are highly electrophilic (Aydin et al., 2023), which can therefore result in higher formation of reactive oxygen species (ROS) and thus an increase in lipid peroxidation (Gill and Tuteja, 2010 ). The increase in TBARS level in our study may be due to the cell producing more ROS. Abdel-Khalek et al. ( 2015 ), the increases in TBARS levels due to the CuO effect of O.niloticus are parallel to our study. Mwaanga et al. ( 2014 ) examined the effect of CuO and ZnO NPs on D. magna and observed that the TBARS level increased. Dovzhenko et al. ( 2022 ), stated that the MDA level increased with the effect of CuO in Mytilus trossulus . Ates et al. 2014 stated that the MDA level increased as a result of CuO exposure in Cyprinodon variegatu s. Aziz and Abdullah, ( 2023 ) stated that the TBARS level increased in L.rohita with the effect of CuO. Riaz et al. ( 2020 ) observed that TBARS levels increased with CuO exposure in L.rohita . Wang et al. ( 2019 ) observed that exposure to Cu NPs increased TBARS levels in Takifugu fasciatus . Dawood et al. ( 2020 ) stated that CuO increased the TBARS level in C. carpio . 5. CONCLUSION After all kinds of pollutants mix with the environment, their final destination is the water environment as a result of various environmental events. It is an undeniable fact that all kinds of pollutants entering the aquatic environment harm aquatic organisms and affect living things including humans by mixing with the food chain. Considering these situations, there is a need to conduct ecotoxicological tests of pollutants such as NPs on aquatic organisms and investigate their effects. As seen in this study supported by literature data, NPs such as CuO cause oxidative stress by damaging cells in various organisms and affect the living existence of the organism. Necessary precautions must be taken to prevent the release of all kinds of pollutants into the environment and aquatic environment. Declarations Declaration of Interest Statement Availability of data and materials Not applicable. The study complies with ethical standards Ethical approval All authors have declared that there is no ethical violation in this article. Consent to participate All authors confirm that the article has not been previously or currently published anywhere. Authors Contributions In laboratory studies, SERDAR O. and ÇiMEN Ç. Işıl,C. has taken place. During the writing phase of the article, AYDIN A.N. and SERDAR O., realized it. Funding The study did not receive any financial support from any institution or organization. Competing interests The authors declare that they have no conflict of interest. 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Çevre bilimi ve kirlilik araştırması 25:12538–12544 Shaw BJ, Handy RD (2011) Physiological effects of nanoparticles on fish: a comparison of nanometals versus metal ions. Environ Int 37(6):1083–1097. https://doi.org/10.1016/j.envint.2011.03.009 Soliman AMH, Ragab MM, S (2018) Oxidative stress and histomorphological markers in the offspring of Poecilia reticulata maternally exposed to metallic and nanoformulated copper. Egypt J Aquat Biology Fisheries 22(2):51–60. 10.21608/ejabf.2018.7981 Speisky H, Gómez M, Burgos-Bravo F, López-Alarcón C, Jullian C, Olea-Azar C, Aliaga ME (2009) Generation of superoxide radicals by copper–glutathione complexes: Redox-consequences associated with their interaction with reduced glutathione. Bioorg Med Chem 17(5):1803–1810. https://doi.org/10.1016/j.bmc.2009.01.069 Srikanth K, Pereira E, Duarte AC, Rao JV (2016) Evaluation of cytotoxicity, morphological alterations and oxidative stress in Chinook salmon cells exposed to copper oxide nanoparticles. Protoplasma 253:873–884. https://doi.org/10.1007/s00709-015-0849-7 Syberg K, Hansen SF (2016) Environmental risk assessment of chemicals and nanomaterials—the best foundation for regulatory decision-making? Sci Total Environ 541:784–794. https://doi.org/10.1016/j.scitotenv.2015.09.112 Tatar S, Çıkıkoğlu Yıldırım N, Serdar O, Yıldırım N, ve Ogedey A (2018) Gammarus pulex 'in Tunceli, Türkiye'. İnsan ve Ekolojik Risk Değerlendirmesi: Uluslararası Bir Dergi 24(3):819–829deki belediye atıksu arıtma tesisinden çıkan ikincil atık suyun ekolojik risk değerlendirmesinde biyomonitör olarak kullanılması Wang T, Long X, Liu Z, Cheng Y, Yan S (2015) Effect of copper nanoparticles and copper sulphate on oxidation stress, cell apoptosis and immune responses in the intestines of juvenile Epinephelus coioides . Fish Shellfish Immunol 44(2):674–682. https://doi.org/10.1016/j.fsi.2015.03.030 Wang T, Wen X, Hu Y, Zhang X, Wang D, Yin S (2019) Copper nanoparticles induced oxidation stress, cell apoptosis and immune response in the liver of juvenile Takifugu fasciatus . Fish Shellfish Immunol 84:648–655. https://doi.org/10.1016/j.fsi.2018.10.053 Vance ME, Kuiken T, Vejerano EP, McGinnis SP, Hochella MF Jr, Rejeski D, Hull MS (2015) Nanotechnology in the real world: Redeveloping the nanomaterial consumer products inventory. Beilstein J Nanotechnol 6(1):1769–1780 Villarreal FD, Das GK, Abid A, Kennedy IM, Kültz D (2014) Sublethal effects of CuO nanoparticles on Mozambique tilapia ( Oreochromis mossambicus ) are modulated by environmental salinity. PLoS ONE, 9(2), e88723 Vutukuru SS, Chintada S, Radha Madhavi K, Venkateswara Rao J, Anjaneyulu Y (2006) Acute effects of copper on superoxide dismutase, catalase and lipid peroxidation in the freshwater teleost fish, Esomus danricus . Fish Physiol Biochem 32:221–229. https://doi.org/10.1007/s10695-006-9004-x Yi Y, Yang Z, Zhang S (2011) Ecological risk assessment of heavy metals in sediment and human health risk assessment of heavy metals in fishes in the middle and lower reaches of the Yangtze River basin. Environ Pollut 159(10):2575–2585. https://doi.org/10.1016/j.envpol.2011.06.011 Yu GB, Liu Y, Yu S, Wu SC, Leung AOW, Luo XS, Wong MH (2011) Inconsistency and comprehensiveness of risk assessments for heavy metals in urban surface sediments. Chemosphere 85(6):1080–1087. https://doi.org/10.1016/j.chemosphere.2011.07.039 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3850318","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266270000,"identity":"b4f9102d-91fe-491e-b22b-625fd97aed9e","order_by":0,"name":"Ayse Nur Aydin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABHUlEQVRIiWNgGAWjYHCDxAaGDwYScvwgdkIBkVoYZ1RYGEs2gLQYEKUlgYGZ50xFosEBEAePFv4Z6Q8fF7bdkzc4ntz2gLdNIsH4/OrEDw8MGOT5xQ5g1SJxI8fYeGZbseGGMw/bDSTbJPLMbrzdLAF0mOHM2QnYrbmRwybN25bAuOFGYpuEYZtEsdmNsxtAWhIMbmPXIn8j/flvoBZ7sBYQ2jzj7OYf+LQY3EgwYwZqSQRrOXBGInEDf+82vLYYnnljLM1zLiF55pmHbZINFRLGEjd4t1kkGEjg9Ivc8fSHn3nKEmz7jqc/k/5jUCfH3392880fFTby/NI4vC8AFVc4ABORAItIYFcOAvxQpfIN6CKjYBSMglEwCqAAAOWxaFvpKqoFAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5657-8958","institution":"Munzur Universitesi","correspondingAuthor":true,"prefix":"","firstName":"Ayse","middleName":"Nur","lastName":"Aydin","suffix":""},{"id":266270001,"identity":"7e141853-a3dd-4c19-904b-a34995acf02b","order_by":1,"name":"Osman SERDAR","email":"","orcid":"","institution":"Munzur University: Munzur Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Osman","middleName":"","lastName":"SERDAR","suffix":""},{"id":266270002,"identity":"00d2e791-3010-46e6-800d-9c50c2c97b50","order_by":2,"name":"Işıl Canan Çiçek Çimen","email":"","orcid":"","institution":"Munzur Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Işıl","middleName":"Canan Çiçek","lastName":"Çimen","suffix":""}],"badges":[],"createdAt":"2024-01-10 12:50:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3850318/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3850318/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49506742,"identity":"40c5b214-6195-4a49-9435-b80d8efd14ac","added_by":"auto","created_at":"2024-01-12 04:13:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19033,"visible":true,"origin":"","legend":"\u003cp\u003eSOD (U/mL tissue) activities of \u003cem\u003eG.pulex\u003c/em\u003e exposed to CuO, different letters above the bar are statistically significant (p \u0026lt;0.05). *Indicates the statistical difference in the same group at different times (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3850318/v1/34eced95b6ade6e392808124.png"},{"id":49506744,"identity":"ddb8340d-8637-4d3e-81e7-746df96b70b7","added_by":"auto","created_at":"2024-01-12 04:13:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18845,"visible":true,"origin":"","legend":"\u003cp\u003eCAT (nmol/min/ml tissue) activities of \u003cem\u003eG.pulex\u003c/em\u003e exposed to CuO, different letters above the bar are statistically significant (p \u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3850318/v1/292591554cfb3a8e598c751c.png"},{"id":49506743,"identity":"8ecc0d9f-49c5-480f-ba78-aa5fa9a7cb74","added_by":"auto","created_at":"2024-01-12 04:13:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17813,"visible":true,"origin":"","legend":"\u003cp\u003eGSH (µM tissue) levels of \u003cem\u003eG.pulex\u003c/em\u003e exposed to CuO, different letters above the bar are statistically significant (p \u0026lt; 0.05). *Indicates the statistical difference in the same group at different times (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3850318/v1/418e5fa2d5f15906b5777df5.png"},{"id":49506745,"identity":"a0c1bcf6-2483-4aab-b114-9bbe1323474d","added_by":"auto","created_at":"2024-01-12 04:13:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16678,"visible":true,"origin":"","legend":"\u003cp\u003eTBARS (µM tissue) levels of \u003cem\u003eG.pulex\u003c/em\u003e exposed to CuO, different letters above the bar are statistically significant (p \u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3850318/v1/300c5670e56d1f5f21de2507.png"},{"id":49740665,"identity":"ed80e44f-0748-41fd-8236-d0d898deb735","added_by":"auto","created_at":"2024-01-17 09:10:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":337041,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3850318/v1/ab8f53f4-5945-4b2a-8d16-23350e53e7bb.pdf"}],"financialInterests":"","formattedTitle":"Determination of Oxidative Stress Responses Induced by Copper Oxide (Cuo) Nanoparticle in Gammarus Pulex","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe exponential growth of nanotechnology has significantly amplified environmental apprehensions regarding the potential ecological hazards associated with engineered nanoparticles (NPs) (Syberg and Hansen, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Extensive nanoecotoxicological research is enhancing our comprehension of the ecotoxicity of nanoparticles (Kahru and Dubourguier, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kunzmann et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Petersen et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Metal oxide nanoparticles (MNPs) are extensively utilized engineering nanomaterials (Vance et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Copper oxide nanoparticles (CuO-NPs) find use in several industries including semiconductor devices, catalysts, photovoltaic cells, wood preservation, textile fibers, biocides, coatings, thermoplastics, colorants, and ceramic additives for optical glass polishing (Ma et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe overabundance of pollutants in aquatic environments leads to substantial environmental and health issues (McNeil and Fredberg, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Pollutants can elevate the concentrations of metals in natural water, leading to significant harm to both freshwater and marine ecosystems (Muhammed et al., 2011; Yu et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; El Nemr, 2012; El Nemr et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The detrimental impacts of heavy metals mostly stem from their capacity to spread and multiply in aquatic food chains, as well as their toxicity and tendency to accumulate in biological tissues (Matta et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; İslam and Tanaka, 2004; Yi et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Copper has considerable toxicity towards aquatic creatures, inducing irreversible harm at concentrations slightly surpassing those necessary for their growth and reproduction (Baldwin et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe accumulation of waste from nanoparticles (NPs) in aquatic species, including fish, bacteria, single-celled organisms, crustaceans, and algae, leads to cellular damage and toxicity (Shaw and Handy \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Nanoparticles (NPs) have been found to accumulate in the organs of aquatic species, and their introduction into water can potentially modify the physiological reactions of these organisms (Chupani et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The presence of CuO-NPs in the aqueous environment can lead to hazardous effects on aquatic organisms. This is mostly caused by the release of copper ions and nanoforms, which the organisms are exposed to (Gomes et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Moreover, both enzymatic and non-enzymatic parameters are commonly employed to forecast the impact of hazardous chemicals on aquatic organisms. The impact of dangerous chemicals on the activity of enzymes is a crucial biochemical factor regulated by stress (Jahanbakhshi and Hedayati \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOxidative stress is a crucial mechanism for protecting against harmful reactive oxygen species (ROS) or healing oxidative damage (Dorval and Hontela \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Copper is a metal that can help protect against pollutants by contributing to antioxidative defense (Parvez and Raisuddin \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Copper can impact oxidative stress due to its potential to enhance the production of reactive oxygen species (ROS) through a Fenton-like reaction, which is a significant factor in lipid peroxidation (Prousek, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). While tiny quantities of copper are necessary for all species and play a crucial function in the regulation of superoxide dismutase, it can be harmful to aquatic animals by depleting glutathione levels (Speisky et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Copper exhibits redox potential and generates an abundance of free radicals, leading to the buildup of O2 and H2O (Moyson et al., 2016; Aliko et al., 2015). Additionally, it serves as a catalyst for Cu, SOD, CAT, and GST enzymes. Typically, the cell's favorable antioxidant and equilibrium are upheld by a mixture of diminutive antioxidant molecules, such as SOD, GST, and CAT enzymes (Glass et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Oxidative stress arises from the impact of heavy metals that disturb the normal functioning of antioxidant enzyme systems (Dimitrova et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Reactive oxygen species that are harmful to the body react with cellular components, resulting in the oxidation of proteins and damage to DNA through oxidation. This process leads to the deactivation of enzymes, tissue damage, rupture of cell membranes, mutations, and ultimately, cell death (Vutukuru et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBioindicators not only provide information about the deterioration caused by the accumulation of various pollutants in ecosystems, but also help determine how long the problems have persisted through physical and chemical tests performed in that ecosystem (Aydın et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Organisms belonging to the Gammaridae family are employed as bioindicators in aquatic habitats (Serdar et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Gammarids are bioindicators that are often present in freshwater ecosystems and are regularly employed in ecotoxicological investigations. These creatures serve as a primary food source for fish and amphibians and are abundant in streams (Aydın et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eG. pulex\u003c/em\u003e has been found to be susceptible to a range of contaminants, as demonstrated by Serdar et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and Tatar et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eG. pulex\u003c/em\u003e was selected as the test organism for this study due to its ecological significance and role in ecosystems. This study aims to analyze the superoxide dismutase (SOD) and catalase (CAT) activity, thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) levels, as well as the absorption amounts of copper oxide nanoparticles (CuO NP), in the bioindicator species \u003cem\u003eG. pulex\u003c/em\u003e. These parameters are indicators of oxidative stress in the aquatic ecosystem.\u003c/p\u003e"},{"header":"2. MATERIAL METHOD","content":"\u003cp\u003e \u003cb\u003eNanoparticles\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNP materials used in the study were obtained from the CuO commercial company (SkySpring). The chemical, which is in the analytical reagent class, was used without any purification or purification. The shape and size data declared by the manufacturer for NP were used in bioassay studies with reference to the shape and size data declared by the manufacturer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOrganism Provision and Adaptation\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eG. pulex\u003c/em\u003e individuals used in the study were collected from the side branches of Munzur Stream in Tunceli province with the help of a bottom scoop and brought to the Munzur University Faculty of Fisheries research laboratory with air supplementation. \u003cem\u003eG. pulex\u003c/em\u003e individuals were placed in 40x20x20 cm aquariums and adapted to laboratory conditions for 4 weeks.\u003c/p\u003e \u003cp\u003eEnvironments suitable for natural habitats were created for the adaptation of \u003cem\u003eG. pulex\u003c/em\u003e to laboratory conditions. For this purpose, sediments taken from the natural environment of G. pulex were washed with pure water and placed in stock aquariums. Water brought from the natural environment of \u003cem\u003eG. pulex\u003c/em\u003e was added to the aquariums. Stock aquariums were supplemented with oxygen using an air engine. A 12:12 dark:light photoperiod was applied in ambient lighting. The ambient temperature was fixed at 18 ͦC with a thermostatic air conditioner and the aquarium water temperature with a chiller. After the adaptation environment was prepared, \u003cem\u003eG. pulex\u003c/em\u003e collected from Munzur Stream were placed in stock aquariums. \u003cem\u003eG. pulex\u003c/em\u003e was allowed to adapt to laboratory conditions. 70% of the water in stock aquariums was renewed weekly. To feed \u003cem\u003eG. pulex\u003c/em\u003e, shrub willow tree leaves were collected and left to rot.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAcute Toxicity Tests and Trial Design\u003c/b\u003e \u003c/p\u003e \u003cp\u003eExperiments have not been conducted to determine sublethal concentrations in order to determine the acute toxicity value, taking into account the harm of pollutants to nature and the environment. Sublethal concentration values were determined by reviewing the literature and appropriate concentration values. In all experimental stages of the research, 0.5 liters of non-chlorinated water taken from the natural environment of the creatures was used in 1-liter glass aquariums. 10 G. pulex were placed in these aquariums for each concentration.\u003c/p\u003e \u003cp\u003eGroup Control; There is no retained CuO concentration in water taken from the natural environment of organisms.\u003c/p\u003e \u003cp\u003eGroup C1; 10 ppm CuO concentration to (CuO),\u003c/p\u003e \u003cp\u003eGroup C2; 20 ppm CuO concentration to (CuO),\u003c/p\u003e \u003cp\u003eGroup C3; (CuO), 40 ppm CuO concentration,\u003c/p\u003e \u003cp\u003eTo determine the changes in oxidative stress and antioxidant biomarker parameters during elimination, samples taken after 96 hours were kept in spring water brought from the living areas for 24 hours, and then the changes in oxidative stress and antioxidant biomarker parameters during elimination were determined.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiochemical analyzes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTissue samples collected at 24 and 96 hours were utilized. The samples were weighed and then mixed with PBS buffer (phosphate-buffered saline) at a ratio of 1 part sample to 5 parts buffer by weight. The mixture was then homogenized using an ice homogenizer in order to assess antioxidant properties. The samples underwent centrifugation at a speed of 17,000 revolutions per minute for a duration of 15 minutes. The resulting liquid portion, known as the supernatant, was then preserved in a deep freezer at a temperature of -86\u0026deg;C until further measurements were conducted. The activities of superoxide dismutase (SOD) and catalase (CAT), as well as the levels of thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH), were measured using ELISA kits obtained from CAYMAN Chemical Company.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSPSS 24.0 package program one-way ANOVA (Duncan 0.05) was used to evaluate biochemical analyses.\u003c/p\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e \u003cb\u003eSOD Activity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e displays the temporal variations of Superoxide Dismutase (SOD) activities in \u003cem\u003eG. pulex\u003c/em\u003e when exposed to various concentrations of CuO. Significant increases in superoxide dismutase (SOD) activities were observed in all concentration groups of CuO at 24 and 96 hours, compared to the control group. The increases were statistically significant, with a p-value of less than 0.05. Significant changes, with a statistical significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, were detected when comparing the application groups with elimination groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCAT Aktivity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e displays the CAT activities in \u003cem\u003eG. pulex\u003c/em\u003e that were subjected to varying concentrations of CuO across different time periods. All groups experienced statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) declines after 24 hours, when compared to the control group. The statistical analysis conducted after 96 hours found that the differences observed between the application groups and the control group were not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Negligible changes (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were seen when comparing the deletion groups and application groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGSH Level\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe levels of glutathione (GSH) in \u003cem\u003eG. pulex\u003c/em\u003e subjected to various doses of CuO at different time intervals are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Significant decreases (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were detected in all amounts of CuO at 24 and 96 hours compared to the control group, according to statistical analysis. Significant changes, with a statistical significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, were detected when comparing the elimination groups with application groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTBARS Level\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e presents the TBARS levels in \u003cem\u003eG. pulex\u003c/em\u003e that were treated to various doses of CuO at different time intervals. Significant increases (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were seen in all groups using CuO at 24 and 96 hours, compared to the control group. Significant changes, with a statistical significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, were detected when comparing the elimination groups with application groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eAquatic creatures, including mammals, have also developed the ROS-scavenging antioxidant defense mechanism. However, any disruption in this system might negatively impact their survival, growth, and physiology (Filho, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Pandey et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The detrimental effects induced by reactive oxygen species (ROS) are counteracted by three prominent antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Superoxide dismutase (SOD) and catalase (CAT) are the main mechanisms that protect cells from oxidative damage (Fang and Zheng, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Superoxide dismutase (SOD) transforms harmful superoxide radicals into hydrogen peroxide (Halliwell and Gutteridge, 2007), which is then broken down by catalase (CAT) into molecular oxygen and water (Chelikani et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In our study, increases in SOD activity and fluctuations in CAT activity are similar to many studies in the literature. Naeemi et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), stated that there were increases in SOD and CAT activity with the effect of CuO NP in \u003cem\u003eCyprinus carpio\u003c/em\u003e. Aziz et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), stated that there were increases in SOD activity and decreases in CAT activity as a result of CuO exposure in \u003cem\u003eHypophthalmichthys nobilis\u003c/em\u003e. Abdel-Latif et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), examined the effects of CuO on \u003cem\u003eOreochromis niloticus\u003c/em\u003e and observed that there were increases in SOD activities. Fazelian et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), stated that CAT activity increased with the effect of CuO on \u003cem\u003eNannochloropsis oculata\u003c/em\u003e. Abdel-Khalek et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), observed increases in SOD and CAT activities due to the CuO effect of \u003cem\u003eO.niloticus\u003c/em\u003e. Srikanth et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), stated that SOD and CAT activity increased with the effect of CuO in \u003cem\u003eChinook salmon\u003c/em\u003e. Aziz and Abdullah, (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) stated that there were increases in CAT activities in \u003cem\u003eLabeo rohita\u003c/em\u003e due to the effect of CuO. Pradhan et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) stated that there were increases in SOD activities in \u003cem\u003eAllogamus ligonifer\u003c/em\u003e exposed to CuO. Wang et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), stated that exposure to Cu NPs increased SOD and CAT activities in \u003cem\u003eTakifugu fasciatus\u003c/em\u003e. Soliman and Ragab (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), stated that SOD activity was increased by CuO in \u003cem\u003ePoecilia reticulata\u003c/em\u003e. Dawood et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) observed that CuO increased SOD and CAT activities in \u003cem\u003eC.carpio\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eGlutathione (GSH) has a crucial function as an antioxidant, preserving the integrity of proteins and enzymes, and acting as a cofactor for the GST enzyme. Several studies have documented a reduction in the levels of glutathione (GSH) in aquatic species following their exposure to metal ions (Loro et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The findings suggests that the decline in GSH level in G. pulex is contingent upon the concentration of metals. The findings of our investigation are consistent with the research conducted by Abdel-Khalek et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which demonstrated a reduction in GSH levels as a result of the impact of CuO on \u003cem\u003eO.niloticus\u003c/em\u003e. Mwaanga et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), stated that CuO and ZnO NPs caused a decrease in GSH levels in \u003cem\u003eD.magna\u003c/em\u003e. Akbary et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), stated that CuO reduced the GSH level on mullet. Srikanth et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), reported that GSH levels decreased in \u003cem\u003eC. salmon\u003c/em\u003e with the effect of CuO. Villarreal et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), stated that decreases occurred in GSH levels due to the effect of \u003cem\u003eO.mossambicus\u003c/em\u003e CuO. Liu et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), found that GSH level decreased in \u003cem\u003eD.magna\u003c/em\u003e with the effect of CuO. Noureen et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that CuO reduced GSH levels in \u003cem\u003eC. carpio\u003c/em\u003e. Wang et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) stated that CuO caused decreases in GSH levels in \u003cem\u003eEpinephelus coioides\u003c/em\u003e. Ogunsuyi et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), stated that GSH levels decreased with the effect of CuO on \u003cem\u003eClarias gariepinus\u003c/em\u003e. Soliman and Ragab (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), stated that GSH level was reduced by CuO in \u003cem\u003ePoecilia reticulata\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eOxidative stress can lead to the degradation of membranes by causing peroxidation of lipid tails in the membrane structure. This process results in the formation of reactive chemicals such malondialdehyde (MDA) (Barata et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). ROS ultimately leads to lipid peroxidation. Lipid peroxidation results in the formation of organic hydroperoxides, which can be broken down into different organic compounds, such as MDA (Barata et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This frequently results in a cyclic detrimental process, since certain byproducts of the breakdown are highly electrophilic (Aydin et al., 2023), which can therefore result in higher formation of reactive oxygen species (ROS) and thus an increase in lipid peroxidation (Gill and Tuteja, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The increase in TBARS level in our study may be due to the cell producing more ROS. Abdel-Khalek et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), the increases in TBARS levels due to the CuO effect of \u003cem\u003eO.niloticus\u003c/em\u003e are parallel to our study. Mwaanga et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) examined the effect of CuO and ZnO NPs on \u003cem\u003eD. magna\u003c/em\u003e and observed that the TBARS level increased. Dovzhenko et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), stated that the MDA level increased with the effect of CuO in \u003cem\u003eMytilus trossulus\u003c/em\u003e. Ates et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e stated that the MDA level increased as a result of CuO exposure in \u003cem\u003eCyprinodon variegatu\u003c/em\u003es. Aziz and Abdullah, (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) stated that the TBARS level increased in \u003cem\u003eL.rohita\u003c/em\u003e with the effect of CuO. Riaz et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) observed that TBARS levels increased with CuO exposure in \u003cem\u003eL.rohita\u003c/em\u003e. Wang et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) observed that exposure to Cu NPs increased TBARS levels in \u003cem\u003eTakifugu fasciatus\u003c/em\u003e. Dawood et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) stated that CuO increased the TBARS level in \u003cem\u003eC. carpio\u003c/em\u003e.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eAfter all kinds of pollutants mix with the environment, their final destination is the water environment as a result of various environmental events. It is an undeniable fact that all kinds of pollutants entering the aquatic environment harm aquatic organisms and affect living things including humans by mixing with the food chain. Considering these situations, there is a need to conduct ecotoxicological tests of pollutants such as NPs on aquatic organisms and investigate their effects. As seen in this study supported by literature data, NPs such as CuO cause oxidative stress by damaging cells in various organisms and affect the living existence of the organism. Necessary precautions must be taken to prevent the release of all kinds of pollutants into the environment and aquatic environment.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe study complies with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e All authors have declared that there is no ethical violation in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e All authors confirm that the article has not been previously or currently published anywhere.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u0026nbsp;\u003c/strong\u003eIn laboratory studies, SERDAR O. and \u0026Ccedil;iMEN \u0026Ccedil;. Işıl,C. has taken place. During the writing phase of the article, AYDIN A.N. and SERDAR O., realized it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThe study did not receive any financial support from any institution or organization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdel-Khalek AA, Kadry MA, Badran SR, Marie MAS (2015) Comparative toxicity of copper oxide bulk and nano particles in Nile tilapia; \u003cem\u003eOreochromis niloticus\u003c/em\u003e: biochemical and oxidative stress. 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Chemosphere 85(6):1080\u0026ndash;1087. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chemosphere.2011.07.039\u003c/span\u003e\u003cspan address=\"10.1016/j.chemosphere.2011.07.039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ds: Gammarus pulex, Copper Oxide, Oxidative stress, Biomarkers","lastPublishedDoi":"10.21203/rs.3.rs-3850318/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3850318/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs Copper Oxide (CuO) has a wide range of uses in industry, it is thought to have a wide polluting effect on the environment and aquatic environment. \u003cem\u003eGammarus pulex\u003c/em\u003e was chosen as the model organism in this study, which was carried out with the aim of realizing the effect of CuO mixed into the water environment. In order to carry out the study, CuO was exposed to 0 (control), 10, 20, 40 ppm concentrations for 24, 96 and 120 (elimination) hours. Samples were taken from the experimental environment at the end of 24, 96 and 120 hours. The study was carried out in 3 replicates. The assessment of oxidative stress and antioxidant biomarkers was conducted using ELISA kits obtained from CAYMAN Chemical Company. The parameters analyzed included the activity of SOD and CAT, as well as the levels of TBARS and GSH. The biochemical studies were evaluated using the one-way ANOVA (Duncan 0.05) feature of the SPSS 24.0 package program.\u003c/p\u003e \u003cp\u003eWith the study data, increases in SOD activities and fluctuations in CAT activity were determined. Decreases in GSH levels and increases in TBARS levels occurred.\u003c/p\u003e","manuscriptTitle":"Determination of Oxidative Stress Responses Induced by Copper Oxide (Cuo) Nanoparticle in Gammarus Pulex","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-12 04:13:05","doi":"10.21203/rs.3.rs-3850318/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be929726-28cb-4eb5-b47b-aefcaf8f945e","owner":[],"postedDate":"January 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-17T09:02:40+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-12 04:13:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3850318","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3850318","identity":"rs-3850318","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}